IAQ5111
EPA
United States
Environmental Protection
Agency
Ofiice oi Health aha
Environmental Assessment
Washington, DC 20460
EPA 500/8-91/202
January 1992
Research And Development
Indoor Air - Assessment
Indoor Biological
Pollutants
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EPA 600/8-91/202
January 1992
Indoor Air - Assessment
Indoor Biological Pollutants
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Enviromental Protection Agency
Research Triangle Park, NC 27711
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ECAO-R-0315
JANUARY 1992
INDOOR BIOLOGICAL POLLUTANTS
Prepared by: Dr. Harriet Burge and
Dr. Thomas Platts-Mills
Environmental Criteria and Assessment Office ;
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Research triangle Park, NC 27711
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
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. FOREWORD
There are significant problems associated with exposure to airborne substances of
biological origin in the indoor environment. It now seems clear that a significant percentage
of the diseases associated with indoor air pollution are related to bioaerosols, and that the
diseases can be more serious, and cause more distress in terms of mortality and morbidity
than those diseases attributed to the common outdoor air pollutants.
This document is intended to represent a few of the known diseases associated with
inhalation exposure to biological aerosols in the indoor environment. This review
summarizes the data available on the nature of bioaerosols and their health effects, methods
of measurement, standards for exposure, approaches toward developing such standards, and
remedial actions. The document is not intended to cover all of the available information on
biological aerosols but instead is designed to be an introduction to a more complete and
comprehensive evaluation of biological aerosols in the indoor environment to follow. An
additional objective is to focus attention on areas of particular importance where little ,or no
research is being conducted.
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PREFACE
In October 1986 Congress passed the Superfund Amendments and Reauthorization Act
(SARA, PL 99-499) that includes Title IVThe Radon Gas and Indoor Air Quality Research
Act. The Act directs that EPA undertake a'comprehensive indoor air research program.
Research program requirements under Superfund Title IV are specific. They include
identifying, characterizing, and monitoring (measuring) the sources and levels of indoor air
pollution; developing instruments for indoor air quality data collection; and studying high-risk
building types. The statute also requires research directed at identifying effects of indoor air
pollution on human health. In the area of mitigation and control the following are required:
development of measures to prevent or abate indoor air pollution; development of methods to
assess the potential for contamination of new construction from soil gas, and examination of
design measures for preventing indoor air pollution. EPA's indoor air research program is
designed to be responsive in every way to the legislation.
In responding to the requirements of Title IV of the Superfund Amendments, EPA-ORD
has organized the Indoor Air Research Program around the following categories of research:
(A) Sources of Indoor Air Pollution; (B) Building Diagnosis and Measurement Methods;
(C) Health Effects; (D) Exposure and Risk (Health Impact) Assessment; and (E) Building
Systems and Indoor Air Quality-Control Options.
EPA is directed to undertake this comprehensive research and development effort not
only through in-house work but also in coordination with other Federal agencies, state and
local governments, and private sector organizations having an interest in indoor air pollution.
The ultimate goal of SARA Title IV is the dissemination of information to the public.
This activity includes the publication of scientific and technical reports hi the areas Described
above. To support these research activities and other interests as well, EPA publishes its
results in the INDOOR AIR report series. This series consists of five subject categories:
Sources, Measurement, Health, Assessment, and Control. Each report is printed in a limited
quantity. Copies may be ordered while supplies last from:
U.S. Environmental Protection Agency
Center for Environmental Research Information
26 West Martin Luther King .Drive
Cincinnati, OH 45268
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When EPA supplies are depleted, copies may be ordered from:
National Technical Information Service
U.S. Department of Commerce
5285 Port Royal Road
Springfield, VA 22161
VI
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CONTENTS
FOREWORD . . . .,. ....... . . . . : .-.' iii
PREFACE .... . .... ..'..; . . ! v
TABLES ..... . . '.'. ... ......... xi
ABSTRACT . . . .... xiii
AUTHORS, CONTRIBUTORS, AND REVIEWERS .'.'.." xv
1. SUMMARY AND CONCLUSIONS ......... . ..'.- ...... . . . . 1-1
1.1 Biological Pollutants ............................ 1-1
1.1.1 Viruses . , . ;~. ' . . .- . . . . 1-1
1.1.2 Bacteria . ... .', .- l-l
1.1.3- Fungi 1-2
1.1.4 Protozoa ...'. 1-2
1.1.5 Arthropods . . 1-3
1.1.6. Mammals and Birds 1-3
1.2 Bioaerosol-Related Disease ........................ 1-4
1.2.1 Infectious Disease . . . . ................. . . . 1-4
1.2.1.1 Human-Source Infections and ' . -
Animal-Source Infections 1-4
1.2.1.2 Environmental-Source Infections . ........ "1-4
1.3 Hypersensitivity Disease > 1-5 .
1.4 Biological Toxins . . .. . . . . 1-6
1.5 Air Sample Collection and"Methods of Analysis 1-7
1.6 Research Needs . 1-8
2. BACKGROUND INFORMATION ^2-1
2.1 INTRODUCTION . . ... ........ . . . . 2-1
2.2 HISTORICAL PERSPECTIVE OVERVIEW 2-2
2.2.1 Infectious Disease ''. . 2-2
2.2.2 Allergies ..".......... . . .-. 2-3
2.2.3 Toxins/Volatiles ..' 2-4
2.2.4 Environmental Control 2-4
3. BIOLOGICAL POLLUTANTS 3-1
3.1 VIRUSES ...... .'......... 3-2
3.1.1 Viral Morphology . . ; .... 3-2
3.1.2 Viral Physiology ......................... 3-2
_3.1.3 Viral Ecology ....... ... ............. .... . 3-3
3.1.4 Diseases Caused by Airborne Viruses 3-3
3.2 BACTERIA 3-4
3.2.1 Bacterial Morphology 3-4
3.2.2 Bacterial Physiology . 3-4
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CONTENTS (cont'd)
Page
3.2.3 Bacterial Ecology 3-5
3.2.4 Diseases Caused by Airborne Bacteria 3-o
3.3 FUNGI I'6
3.3.1 Fungal Morphology « ;f °
3.3.2 Fungal Physiology *~'
3.3.3 Fungal Ecology Y1
3.3.4 Diseases Caused by Airborne Fungi . J-o
3.4 PROTOZOA *~*
3.4.1 Protozoan Morphology , ?* V ' ;: ~*
3.4.2 Protozoan Physiology % ^-9
3.4.3 Protozoan Ecology f"*
3.4.4 Diseases Caused by Protozoa . 3-9
3.5 ARTHROPODS ^
3.5.1 Mites - 5~\
3.5.2 Cockroaches ; *
3.5.3 Other Arthropods 6~l L
3.6 MAMMALS AND BIRDS 3'u
4. BIOAEROSOL-RELATED DISEASES 4-1
4.1 INFECTIOUS DISEASE *-l
4.1.1 Human-Source Infections 4~^
4.1.2 Environmental-Source Infections 4'6
4.1.3 Animal-Source Infections 4-11
4.2 HYPERSENSinvrrY DISEASE 4-11
4.2.1 Rhinitis, Asthma, and Allergic Bronchopulmonary
Aspergillosis 4-12
4.2.1.1 Causative Agents . - 4'14
4.2.1.2 Diagnosis of Immediate Hypersensitivity ..:. 4-18
4.2.2 Hypersensitivity Pneumonitis . . . . 4-^
4.2.2.1 ' Causative Agents 4-19
4.2.2.2 Detection of Sensitization 4-22
4.3 BIOLOGICAL TOXINS 4'^
4.3.1 Bacterial Toxins 4'^j
4.3.2 Mycotoxins 4-25
4.3.3 Fungal Volatile Organic Compounds 4~//
5. BIOAEROSOL INVESTIGATIONS - 5-1
5.1 Investigative Strategies . .
5.1.1 Studying Indoor Microbial Ecology with
Respect to Air Pollution 5-2
5.1,2 Documenting Exposure/Dose/Symptom
Relationships ' ; 5"2
Mil
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CONTENTS (cont'd)
5.1.3 Documenting Unusual Exposure Situations .... . .... - 5-2..'
5.2 AIR SAMPLE COLLECTION TECHNOLOGY ........... 5-3
5.2.1 Principles of Aerosol Sampling 5-4
5.2.2 Sampling for Viable Microorganisms ..... ... . . . . . . 5-6
5.2.3 Sampling for Microscopically
Identifiable Organisms ...................... 5-8
5.2.4 Sampling for Amorphous Particles ... ........ 5-8
5.2.5 Sampling for Volatile Aerosols ................ 5-9
5.2.6 Source Sampling '. 5-9
5.3 AIR- AND'SOURCE-SAMPLE ANALYSIS METHODS ...... 5-10
5.3.1 Direct Microscopy 5-11
5.3.2 Culture ? 5-12
5.3.3 Immunoassay 5-14
5.3.4 Bioassays and Chemical Analysis 5-16
6. CONTROL OF BIOAEROSOL-INDUCED DISEASE 6-1
6.1 BUILDING DESIGN ................ 6-1
6.2 BUILDING MAINTENANCE 6-2
6.3 REMEDIAL ACTIONS .......................... 6-2
7. REFERENCES .......... ... 7-1
IX
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TABLES
Number Page
3-1 Biological Pollutants . ..... ........ 3-1
3-2 Morphological Characteristics of Some .
Common Bacteria ', 3-4
4-1 Aspergillus Species Implicated in Cases
of Infectious Disease .. 4-10
S " " ' - .. ' .
5-1 Summary of Sampling Modalities Useful
for Indoor Bioaerosols . " 5-11
XI
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ABSTRACT
Biological aerosols have been recognized as indoor hazards for several hundred years.
Pasteur demonstrated that infectious diseases are transmitted through indoor air. Dust has
« ... '
been a recognized allergen since the mid-19th century. Recently, however, the role of indoor
air in transmission of infectious disease has been de-emphasized, and the problems associated
with other kinds of indoor bioaerosols have received only minimal public health attention.
This is in spite of the fact that we spend an average of 22 hours/day indoors. Influenza
causes 10,000 deaths per year. The house dust mite is probably the single most important
cause of asthma among children and young adults. Indoor allergens are thought to be
responsible for as much as 50% of the incidence of acute asthma in adults under 50 years
old. Microbial toxins are among the most toxic substances known to man with effects that
include acute toxicity symptoms, birth defects, cancer, and, in some cases, death: The
concentrations and health effects of these toxins are completely unknown for the vast majority
of indoor environments. Volatile organic compounds are produced by all microorganisms
and accumulate in confined spaces, causing odors and possibly unknown health effects. The
nature of these substances, their health effects, and concentrations in indoor environments is
unknown. .
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
The principal authors of this document are Dr. Harriet Burge; University of Michigan,
"-,'-" " .' *
and Dr. Thomas 'Platts-Mills, University of Virginia. This document was prepared under
contract with 'the Environmental Protection Agency's Office of Health and Environmental
Assessment (OHEA), Environmental Criteria and Assessment Office (ECAO). The ECAO,
Indoor Air Research Program, had the overall responsibility for coordination and preparation
of this document (Beverly M. Comfort, Project Manager).
The following individuals provided peer reviews of this document:
Michael A. Berry
U.S. Environmental Protection Agency
Environmental Criteria and Assessment
Office
Research Triangle Park, NC
Arthur Chiu
U.S. Environmental Protection Agency
Human Health Assessment Group
Washington, DC
Sheila Rosenthal -
U.S. Environmental Protection Agency
Human Health Assessment Group;
Washington, DC
Chin S. Yang
Federal Employee Occupational Health
3585 Market Street
Philadelphia, PA
William Ewald
U.S. Environmental Protection Agency
Environmental Criteria and Assessment
Office
Research Triangle Park, NC .
Harriet Ammann
Washington State Department of Health
Office of Toxic Substances
Olympia, WA
Steve Bayard
U.S. Environmental Protection Agency
Human Health Assessment Group
Washington, DC
Robert Dyer
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC
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1. SUMMARY AND CONCLUSIONS
1.1 BIOLOGICAL POLLUTANTS
Biological pollutants are agents that are derived from or are living organisms. These
agents include viruses, bacteria, fungi, protozoa, and their toxins and arthropod, mammal,
and bird antigens.
1.1.1 Viruses
Viruses are the smallest of all life forms and contain either RNA or DNA. They are
heterogeneous and vary in size, morphology, chemical composition, host range, and host
effects. Viruses have no physiology of their own, but rather are obligatory intracellular
parasites that mobilize host cell processes and lack the ability to reproduce on their own.
Viruses enter the host through the skin via animal bites or open wounds, through the
respiratory tract, the alimentary tract, and the urogenital tract. Those entering through the
respiratory tract must be able to survive in air. ,
Airborne viruses almost always require the presence of someone with an active infection
in a state "that includes coughing or sneezing and less commonly, infected animals housed
indoors. Factors affecting viral survival in air include temperature, relative or absolute
humidity, the presence of ultraviolet light, the nature and size of the particle to which the
virus is attached, and possibly the presence of other pollutants. Some of the most common
diseases produced by viruses are influenza, some common colds, meailes^trubeola), chicken
pox, and rubella (German measles). ""'r
1.1.2 Bacteria
Bacteria are prokaryotic (have no true nucleus) microorganisms characterized by a rigid,
polyfflccharide-rich cell wall; a single chromosome unbounded by a nuclear membrane; and
no mitochondria. Bacteria are classified by cell shape (spherical, rod shaped, filamentous)
and arrangement (single, chains, clumps, pairs, tetrads), by reaction to the Gram stain, and
by biochemical and physiological reactions. Almost all bacteria require a source of
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carbohydrate. Most bacteria utilize nonliving organic matter and are called saprophytic or
saprophytes. Bacteria utilizing living tissue are known as parasites or are parasitic. Bacteria
able to utilize both living and nonliving matter as an energy supply are facultative parasites
and those bacteria that survive only on living tissue are known as obligate parasites.
To transmit infectious disease, bacterial cells must remain viable in air. The factors
affecting their survival are similar to those for viruses. Some bacteria produce spores that are
highly resistant to environmental damage and are difficult to kill even with biocidal materials.
Aerosol-transmitted diseases caused by bacteria include infectious diseases, hypersensitivity
diseases, and toxicoses.
1.1.3 Fungi
Fungi are eukaryotic microorganisms with cells containing one or more organized nuclei
as well as other membrane-bound organelles. Fungi may be unicellular or multicellulaf and
reproduce mainly by spores. Formally, fungi are classified into two groups based on sexual
reproduction: Zygomycetes (characterized by a resting' zygdspore) and Dikaryomycetes
1 ' ' " ' * .
(characterized by a binucleate multicellular stage preceding nuclear fusion). Most of the
important fungi that are associated with indoor air quality are in the Dikaryomycetes group.
The primary source of indoor airborne fungal spores is the outdoor air. Water is the -
single most important factor in determining whether saprophytic fungi will be found and
survive in a given indoor environment.
Most fungi are saprophytic. There are, however, some facultative parasitic fungi and a
. few obligate parasites. ~ Some fungi and their metabolic by-products have had a major impact
on humans. Antibiotics and mycotoxins are fungal by-products and fungi are used in the
production of some food items. Airborne fungi are responsible for some infectious diseases,
hypersensitivity diseases, arid toxicoses.
1.1.4 Protozoa
Protozoa are primarily unicellular organisms that can live wherever water and nutrients
are of sufficient quantity to support life. Many protozoa are parasitic. Some protozoa
(amoebae) are capable of ingesting gram-negative bacteria, which remain alive within the '
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organism protected from environmental stresses. Two genera of the amoebae (Naegleria and
Acanthamoeba) have been implicated in indoor air-related hypersensitivity disease..
1.1.5 Arthropods
Arthropods include mites, cockroaches, crickets, house flies, moths, and a variety of
beetles. Many different species of mites are found in the home. Live house dust mites are
found deep in carpets, furnishings, and bedding. The mite population of a home is closely
related to the relative humidity in the house; the higher the humidity, the greater the mite
population. The major source of food for mites is human skin scales; however, mites also
depend on fungi for growth.
Cockroaches are present in many homes and can increase to overwhelming numbers if
not controlled. Mites and cockroach allergens are suspected of causing.hypersensitivity
reactions in asthmatics.
1.1.6 Mammals and Birds
Many different mammals and birds are kept in the home and work environment. It is
estimated that approximately 100 million-domestic animals reside in homes in the United
States today. The most common of the domestic animals is the cat. All of these animals shed
proteins and occasionally bacteria or viruses into the environment. Animal effluent can cause
respiratory allergies and, in rare cases, infectious disease.
In most parts of the world, dogs are of much less importance than cats as the cause of
asthmatic attacks. Possibly 10% of all acute asthma in young adults is-rekted to cat allergen
exposure. Laboratory-animal allergy has become a serious occupational"problem. Also,
individual cases of sensitization to pet rodents, producing rhinitis or contact urticaria, have
been documented. Sources of allergens include skin scales, saliva, urinary proteins, serum,
and feathers.
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ll2 BIOAEROSOL-RELATED DISEASE
1.2.1 Infectious Disease
The potential for transmittal of an infectious microorganism in air is first dependent
upon the presence of the organism in the environment. The microorganism must also be able
to survive and multiply in the environment, become airborne in sufficient concentration, and
remain viable long enough to cause disease. For infection to occur the microorganism must
be virulent. Once airborne, the microorganism must come in contact with a susceptible
human host. The susceptibility of the human host is related to the immune status of the host.
Factors that damage the immune system will increase the risk of infection in the exposed
person.
1.2.1.1 Human-Source Infections and Animal-Source Infections
Human-source infections or diseases usually rely on the human host to function as a
reservoir, amplifier, and/or disseminator. These aerosol-transmitted diseases are generally
respiratory infections whose symptoms include coughing and sneezing. Airborne human-
source diseases rarely occur outdoors because of the large mass of air available to dilute the
aerosol and because of environmental factors hostile to microorganisms.
The. human-source infections that are currently considered important with respect to
indoor air quality are influenza, common colds associated with some viruses, and
tuberculosis. Other aerosol-transmitted diseases include measles, rubella, and chicken pox:
Chicken pox is probably the most contagious of these aerosol-transmitted diseases. However,
the measles virus is so virulent that only :4 infectious units/minute released from an infected
host can initiate an epidemic.
Under certain circumstances some microorganisms usually restricted to animal species
may infect humans. The best known of these diseases are Q-fever, anthrax, and brucellosis;
however, the incidence of these diseases is probably low.
1.2.1.2 Environmental-Source Infections
Environmental-source infections result from exposure to inanimate reservoirs
contaminated primarily with saprophytic organisms.
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The primary fungal pathogens (Histoplasma, Coccidioides, and Blaswmyces) grow and
reproduce in nature as soil saprophytes. These saprophytes produce mycelium and spores as
fungi do. When the spores gain access to the human respiratory tract they can produce
disease. The opportunistic saprophytic pathogens normally occupy natural environments and
cause infectious disease only when they penetrate susceptible human hosts. Susceptibility
requires some lack of immune system function. Some of the most common opportunistic
pathogens that cause air-borne disease are the bacteria Legionella pneumophila,
Pseudomows, and Adnetobacter. The best known opportunistic fungal pathogen is
Aspergittus fianigants. This organism produces toxicoses and allergies and. occupies both
natural and manmade environments. >....
1.3 HYPERSENSITIV1TY DISEASE
The hypersensitivity diseases are caused by individual immunologic sensitization to
specific antigens. Antigens are able to stimulate production of antibody or antigen reactive
cells and serve as specific targets for the antibody or sensitized cell. Proteins, lipoproteins,
glycoproteins, polysaccharides, lipopolysaccharides, larger polypeptides, and nucleic acids are
all potential antigens. There are three forms of immune response to indoor air biological
contaminants (antigens): immunoglobulin E (IgE) antibody response (immediate allergic
response); immunoglobulin Q (IgG) antibody response (only detected through serum
immunoassays); and T cell response (delayed allergic response).
The hypersensitivity diseases most clearly associated with indoor airquality are asthma,
rhinitis, and hypersensitivity pneumonitis. Asthma and rhinitis produce^aii IgE antibody
response (immediate) and a T cell response (delayed). It is estimated that 30 to 45% of acute
asthma in children over 7 years old and in adults under 50 years of age can be attributed to
indoor allergen exposure. For some of the indoor inhaled allergens, the relationship to a
disease is obvious to the patients (e.g., cat allergens where the onset of rhinitis, asthma, or
conjunctivitis follows within 15 minutes of entering a house with a cat). Other allergen-
related hypersensitivity diseases are not as obvious, requiring challenge studies with specific
allergens and or epidemiological studies on random populations. Biological agents known to
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produce antigens that cause allergic rhinitis and asthma include fungi; algae; higher plant
spores (pollen) and other parts used as food; and arthropod, avian, and mammalian effluent.
Hypersensitivity pneumonitis is an IgG antibody response and T cell response. It is one
of the most serious of the building-related illnesses. This form of T cell response differs
from that of asthma and rhinitis, and is believed to be caused by some bacteria, fungi,
protozoa, and bird and mammalian serum proteins. .Because the disease is not expected to
occur in "so-called" clean environments (offices, homes) and in early stages misdiagnosis is
probably common, the actual incidence of hypersensitivity is unknown. Until the antigen
source is removed, sensitized individuals cannot return to that environment. Progressive,
irreversible lung damage may occur with continued exposure to the antigen.
1.4 BIOLOGICAL TOXINS
i .,-.
Biological toxins may enter the mammalian system by ingestion; absorption through the
skin; inhalation; and subcutaneous, intraperitoneal, or intravenous injections. Toxic effects
can be acute and/or chronic'. The biological toxins are mainly cytotoxic; however, many are
also teratogenic, mutagenic, and/or carcinogenic. The biological toxins important in indoor
air are bacterial toxins, mycotbxins, and fungal volatile organic compounds.
Bacteria produce both exptoxins and endotoxins; The exotoxin produced by Clostridium
botulinwn is responsible for the potentially lethal form of food poisoning known as botulism.
The role Of airborne exotoxins have not been studied to any great extent. Endotoxins, known
as "fever inducers", are a component of the outer membrane of grariirnegative bacteria that
cause acute pulmonary changes and local inflammatory responses in exposed individuals.
Mycotoxins (fungal metabolites) have a range of toxic effects from mild acute toxicity
to potent carcinogenicity. Mycotoxins enter the body through the skin, gastrointestinal tract,
or respiratory tract. Generally, the mycotoxins are cytotoxic. Some mycotoxins are toxic
without metabolic conversion, whereas others require metabolic conversion to exert their
toxic effects. Those requiring metabolic conversion usually affect the organs where the
metabolism takes place, such as the liver.
All organisms produce and emit volatile organic compounds (VOCs) during growth.
The effects of these volatile organics are generally restricted to annoying odors (smelly socks,
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body odor); however, some compounds may produce significant adverse health effects. It
has been suggested that fungal VOCs may contribute to some cases of "sick building
syndrome" or produce symptoms which mimic that condition.
1.5 AIR SAMPLE COLLECTION A3MD METHODS OF ANALYSIS
Air sampling can be done with the use of personal samplers or by using ambient
monitoring equipment (centrifugal force, inertia! impaction, filtration, and electrostatic
impaction devices). The most commonly used types of equipment are themertial impactor
and filtration devices.
When choosing a sampler for collection of a viable aerosol, the size of the particles, the
relative fragility of the organisms, and the expected concentrations must be considered.
Some organisms must remain viable during sampling and analysis for identification.
Therefore, the media used for collection should not adversely affect viability. Culture plate
impactors, liquid impingers, membrane filter cassettes, and high-volume electrostatic devices
have been used to collect viable microorganisms. Viruses and infectious bacteria, fungi, and
protozoa are usually sampled by methods that allow their culture both in vitro and in vivo.
For very small particles (small fungus spores, thermophilic actinomycetes), suction devices
and isoldnetic samplers should be used. Once the sample -has been collected, it may be
examined by direct microscopy without prior staining (fungal spores) or with prior staining
(bacteria).
Commonly used methods for sampling aerosols for. microscopic idjntiftaaticm are by
suction slit impactors, membiane filter cassette samplers, and rotating agfimpactors.
Suction slit impactors overestimate small aerosolized particles in still aif and underestimate
them in moving air. However, samples can be collected over long periods of time for,
analysis using these devices. The most commonly used samplers in the United States for
outdoor bioaerosol collections are the rotating arm impactors.
Many of the allergens, antigens, and toxins that accumulate in the indoor environment
are carried on particles that do not grow in culture and are not readily identifiable
microscopically. Collection of these particles from air must enable the particles themselves
or the soluble adhering material to be eluted for assay or assayed directly from the sampling
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medium. For water-soluble materials that are not hydrophobia, liquid impingement may be
used. For endotoxins, smooth surface polycarbonate filters minimize the risk of permanent
adherence of the toxins to filter surfaces. Limulus (LAL) bioassays are necessary to
3 '".""" '
determine the biological activity of the endotoxin. High-volume filtration devices have been
used for mite;, cat, and small mammal antigen collection. Proteins derived from these
sources can only be measured by immunoassay.
1.6 RESEARCH NEEDS
The quest for knowledge related to indoor bioaerosols is essentially a quest for a means
to control the diseases they cause. Although we1 have made some progress in collecting the
existing knowledge that applies to~this quest, very little research has been specifically directed
toward indoor biological aerosols, and gaps remain that prevent an accurate assessment of the
risk to human health imposed by these aerosols. The following are needed to more
adequately understand the role of biological aerosols in the indoor environment and health.
Field tests of available bioaerosol instruments for variability, sensitivity, and reliability.
Experimental determinations of statistically appropriate sampling strategies.
Development of integrated sampling devices for viable aerosols.
Development of .single instrument sample collection devices susceptible to multiple analysis
approaches. ^ .
An in-depth evaluation of the role of ventilation in the spread of influenza and other
airborne respiratory infections and the effects of dilution ventilation on the spread of such
diseases. A relatively straightforward epidemiological approach may be appropriate.
Dose-response relationships for environmental exposures. Until these relationships have
been established at least for the most common and important bioaerosols, guidelines for:
determining safe levels cannot be set. These studies will require sophisticated chamber
research, as well as collection of baseline air prevalence data, and carefully designed
investigations that include air sampling as well as epidemiology of specific epidemics of
. bioaerosol-related disease.
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.«*
Baseline data on airborne prevalence for common and important bioaerosols. These data
are essential for establishing rational guidelines and for the development of dose-response
relationships. Adequate equipment is currently available to begin this effort on a
nationwide basis.
Real-time sampling methods that will allow analysis of multiple types of bioaerosols from
a single sample.
Immunological and biochemical assays for a wide range of the most common bioaerosols.
For many, basic research on the nature of antigens and/or toxins, and development of
specific monoclonal antibodies, will be necessary.
, Time-discriminating methods for sampling viable microorganisms. At present, only short
term (minutes) grab samples are possible, making mapping of changing viable aerosol
concentrations cumbersome. Immunological assays do not substitute for these cultural
sampling methods. Immunological assays do not assess viability, information required for
pathogen exposures. ' '
. Evaluation of complaint environments for multiple bioaerosols as well as other pollutants.
It is becoming clear that bioaerosols interact with each other and with other air pollutants
to cause health effects. In particular, ehdotoxin appears to be important in connection
with exposure to sensitizing agents, and environmental tobacco smoke may exacerbate
health effects from a variety of infectious, antigenic, and toxic bioaerosols. In addition,
carefully designed chamber studies using animal models as well as people will be
necessary.
. Examination of antigenic cross-reactivity patterns need to be examined for the most
common environmental fungi and identification of widely prevalent (common) fungal
antigens that can be used to make monoclonal antibodies for clinical assessment and in
assays of air samples. These studies require the interaction of mycologists experienced in
"aeromycology" and immunologists experienced in developing immunoassays and
monoclonal antibodies.
. Characterization of microbial volatile organic compounds with respect to sources, factors
controlling production, prevalence in the environment, and health effects. Characterization
will require choosing appropriate strains of common environmental microorganisms and
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designing studies to evaluate conditions controlling VOC production as well as
identification of VOCs produced. ,
An assay system, possibly based on monoclonal antibodies, that will quickly detect specific
toxins from both dust and air samples. In addition, the many fungi common in indoor
environments should be studied for toxin production, concentrations of specific toxins
estimated with respect to spore levels, assays designed to measure specific new toxins, and
assessment of health effects of these compounds.
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2. BACKGROUND INFORMATION
2.1 INTRODUCTION
Most Americans spend the majority of their lives in the indoor environment. For
many, the time spent indoors can be as long as 22 hours/day (Spengler and Sexton, 1983). It
is, therefore, not surprising that the quality of indoor air is at least as important to health as
the quality of outdoor air. Considerable effort over the last decade has focused on outdoor
air pollution. Research on outdoor air pollution has-focused on establislung risk levels or
* ., , . , , , , , - , v
exposure limits for many of the known hazardous nonbiological pollutants (Yocom et al.,
1971). Most research on indoor air quality has focused on the same pollutants that are found
in outdoor air (e.g., nitrogen oxides, carbon monoxide) and a few other nonbiological agents
that are susceptible to easy measurement (e.g., asbestos, radon), or have stimulated major
controversy (environmental tobacco smoke). It is important to note that research on
nonbiological pollutants is often designed to demonstrate whether or not measurable health
effects occur, whereas research on bioaerosols focuses on methods for measurement of
pollutants that have been known to cause serious health effects for hundreds of years.
Biological aerosols have been the subject of few coordinated research efforts designed to
measure risk or to establish standards for exposure. However, the role of indoor exposure in
the spread of infectious diseases, including tuberculosis, influenza, measles, and whooping
cough, has been known for years. Some information is also available on the dose required to
cause these diseases (Knight, 1980). ^^
Over the last 20 years, there has been a steady, increase in knowleligfof the biological
sources that give rise to immunological sensitization.' This information has not only identified
specific health risks, but has established methods for measuring some of the agents in homes
that are thought to cause hypersensitivity disease. For a few sensitizing agents, it has been
possible to propose specific levels of exposure that represent a risk for sensitization and for
symptom development (Plate-Mills and Chapman, 1987), but for most, insufficient data are
available to propose risk or threshold levels. No research has been directed to identifying the
health effects, prevalence, or relative risks of toxins and volatile irritants of biological origin
in indoor air.
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2.2 HISTORICAL PERSPECTIVE OVERVIEW
Throughout the early days of human existence, hygienic considerations were of low
priority. Although attempts were made to control bacterial or fungal damage to food supplies
(i.e., by drying or salting), little attention was applied to the condition of the house. Floor
coverings often consisted of straw or dirt, and refuse (including human waste) was allowed to
accumulate either within or immediately adjacent to indoor environments. Given our
knowledge of mite and fungal growth, it seems inevitable that materials in these houses Dotted
and became infested with mites and vermin. In fact, rats, mice, and arthropod infestations
were continuing problems that were addressed onlywhen food supplies were threatened.
Stately European houses of the 16th and 17th centuries became so foul after 6 months that it
was common practice to move to allow cleaning. At least partly as a result of these indoor
conditions, the average life expectancy remained near 25 years, and disease (e.g., whooping
cough, smallpox, plague, tuberculosis) regularly decimated major population centers well into
the 19th century. , , ,
2.2.1 Infectious Disease
; Gregory (1961) presents a fascinating history of the discovery of germs and their -
connection with human disease.. He points out that Hippocrates felt epidemic fevers were the
result of inhalation of air infected with pollutants hostile to the human race.. According to
Gregory (1961), Lucretius hypothesized, from observations of the movement of dust motes in
a sunbeam, the existence of what he termed "atoms" that carried disease. It was, however,
not until the 17th century that Leeuwenhoek developed hand-made lenses that allowed
bacteria to be seen for the first time. He described yeasts, infusoria, and a mold. That these
microscopic creatures actually caused disease was not established until the mid 19th-century.
The last 25 years of the 19th century were considered the golden age of bacteriology. Koch's
postulates describing the steps necessary to establish the cause-effect relationship between an
agent and a disease were published in 1878. Before 1900, the microbial agents responsible
for cholera, tetanus, the black plague, leprosy, gonorrhea, and tuberculosis had been found
and viruses had been discovered (Gregory, 1961).
In 1873, Cunningham attempted to collect the agent causing cholera from the air of
jails. He found many fungus spores and pollen grains, but found no correlation between the
' 2-2
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agents he saw and the incidence of cholera. In fact, most disease-producing bacteria are
relatively fragile in the airborne state, and can rarely be isolated. This problem continues
today. For example, Legionella has not been isolated from air in any quantitative fashion,
and establishing cause-effect relationships between specific sources and disease is dependent
on hypothetical dispersion from the source (Muder et al., 1986). Because of these sampling
problems, demonstration of the role of air in the transmission of infectious disease has
focused on elimination of any other form of contact between the infected and noninfected
person. Unequivocal evidence now exists that, among others, influenza, some forms of the
common cold, measles, chicken pox, tuberculosis, anthrax, Q-fever, brucellosis, and a
variety of fungal infections are transmitted via the airborne route (Surge, 1989b).
V
2.2.2 Allergies
Episodes of disease and demise, now recognized as allergic reactions, have been
recorded for over 5,000 years. For example, King Menes of Memphis died in about
3000 B.C. either of anaphylaxis from the sting of a hornet, or was trampled by a
hippopotamus (hornet and hippo sharing the same ancient Egyptian word). During the 16th,
17th, and 18th centuries, reactions were noted, mostly from foods, that were surely allergic
in nature and some surprisingly intuitive observations were made regarding cause and effect.
During these centuries, cats, dogs, horses, feathers, and many foods were observed to cause
asthma. In the early 19th century, it was recognized that pollen caused hay fever, and that
dust from beaten carpets produced similar symptoms.
The first mold allergies were reported in 1924, and it was recognized that damp moldy
,..._.' .Tdi;^*?r.y
homes were conducive to asthma. Mites were observed in house dustjyhe 17th century,
and in the 18th century it was recognized that dust caused asthma. In the 1920s, mites began
to be suspected as the cause of house dust asthma. The first concrete evidence that patients
could be specifically sensitized to house dust came in the 1920s when wheal and flare
responses produced by skin testing with extracts of dust from the homes of sensitized
individuals were reported by Kem (1921). At that time, it was already known that cats,
horse hair, and molds could give rise to this form of sensitization. Experiments to identify
the house dust allergen continued until 1964 when Voorhorst and his colleagues in Holland
2-3
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demonstrated the importance of dust mites of the genus Dermatophagoides (Voprhorst et al.,
1967, 1969).
In the time since, much progress has been made toward recognizing specific causes of
asthma and hay fever, and in defining the mechanisms by which symptoms are elicited. The ^
wheal and flare skin test was developed for the diagnosis of allergies. In 1967 the Ishizakas
established that wheal and flare sTrin responses are mediated exclusively by antibodies of the
IgE isotype. Thus, it is now known that much of the acute sensitivity to allergens can be
explained by the production of IgE antibodies specific for proteins and glycoproteins derived
from living organisms.
2.2.3 Toxins/Volatiles
Ergotism, a devastating disease caused by ingestiori of a mycotoxin, was described by
the Spartans in 430 B.C. The disease results in loss of peripheral circulation, gangrene, and
death. Epidemics during the Middle Ages were known as St. Anthony's Fire because
sufferers prayed to SL Anthony for relief. During the last 30 years, more than
200. mycotoxins have been discovered in 150 different fungi and more are characterized each
year. Nearly all mycotoxin research has centered on ingestion and diseases of animals.
(Kendrick, 1985).
The odor associated with fungal growth is caused by the release of volatile organic
compounds. These compounds vary depending on the substrate being used by the fungus.
A case of arsenic poisoning was described in 1891 that was ultimately connected to fungal
volatiles. Arsenic compounds in wall-paper pigment were transformed to trimethyl arsine by
the action of fungi (Foster, -1949). Remaining research has centered on the use of volatile
compounds as an aid in fungal taxonomy (Halim et al., 1975) and the identification of fungal
volatiles in foods (Kaminski et al., 1974).
2.2.4 Environmental Control
Attempts to isolate infected people and increase ventilation were made when it was
discovered that diseases such as tuberculosis could be transmitted through the air. The very
high ceilings and tall windows of public buildings and prosperous homes of the 18th and 19th
century were an attempt to provide ventilation to reduce human-source aerosols (both
: ' ' ' ' '.''' 2-4 - ' - .
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3.1 VIRUSES
3.1.1 Viral Morphology
Viruses are the smallest of all biological entities. They are a heterogeneous group of
a-ents that vary in size, morphology, chemical composition, host range, and host effects
(American Conference of Governmental and Industrial Hygienists, 1989b). All are
submicroscopic 0*. cannot be resolved with the light microscope). They range in s,ze from
20 to 300 run and have a nucleic acid core (either RNA or DNA) and a protein coat. Some
are enclosed in a lipoprotern capsule. The complete virus particle is known as the
virion.
3.1.2 Viral Physiology
Viruses have no physiology of their own, but rather mobilize host .cell processes. All
are obligate intracellular parasites that lack the genetic information necessary for the synthes,s
of cellular systems. They use host cell metabolic pathways and ribosomes to power thar
' reproductive cycle. Viruses interact with cells in several ways: They may invade the cell
and produce no obvious effects; cause cell lysis and death; or become integrated in the host
cell DNA, altering the cell's genetic makeup.
The actual life cycle of viruses involves several steps. First, the virus is absorbed or
attaches to the cell surface of the host. The host cell is then penetrated by the entire vmis
p^cle or just the vita! genetic material. Prior to replication, the viral nucleic acid separates
from the coating materials. The genetic material then divides and new virus particles are
formed and released. ,^^. %Hl .
Viruses can enter "the host through the skin via animal bites or operands; the
respiratory tract, with ^ site of deposition depending on the size of the particle carrymg the
viruses- the alimentary tract; and the urogenital tract. Although many viruses primanly enter
through a single pamway, most can probably use all portals of entry. Those mat primanly
eater by the respiratory traa via air must be able to survive in air. Many viruses that are not
usually contracted from airborne exposure are fragile and do not live for long outs.de the
protective host environment.
' Viruses undergo evolutionary change, sometimes rather rapidly. Immunological _
methods designed to protect against specific diseases require recognition of specific viral
3-2
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characters and become ineffective over time. Viruses for which this phenomenon is
/
especially evident include influenza and human immunodeficiency virus (HIV) (the acquired
immunodeficiency syndrome [AIDS] virus).
3.1.3 Viral Ecology
As stated above, all viruses are obligate intracellular parasites and, therefore, never
grow or reproduce in environmental reservoirs. Thus, sources (humidifiers, cooling towers)
that may harbor bacterial arid fungal infectious agents are never sources for viral infections.
Although some viruses are relatively hearty and survive for hours or days in air and dust, it
is important to remember that transmission of airborne virus disease almost always requires .
the presence of someone with an active infection in a stage that includes coughing or
sneezing.
Factors affecting viral survival in air include temperature, relative or absolute humidity,
ultraviolet light, and possibly other factors such as the presence of other pollutants. In '
addition, the nature and size of particles on which viruses are carried are important factors in
viral survival (Goodlow and Leonard, 1961; Buckland and Tyrrell, 1962; Gerone et al.,
1966; Karimetal., 1985).
3.1.4 Diseases Caused by Airborne Viruses
Diseases caused by airborne viruses include influenza, the common cold, measles
(rubeola), chicken pox, rubella (German measles), and other less common entities. Viruses
are infectious agents and do not cause hypersensitivity disease or toxic syndromes... There are-
animal viruses, plant viruses, and bacterial viruses. Within each class, individual viruses are
usually specific for one or a small group of hosts, recognizing specific sites on the
appropriate host cell. Thus, most animal viruses do not infect humans. Exceptions, of
course, do occur. Rabies, for example, attacks a wide range of mammals.
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3.2 BACTERIA
3.2.1 Bacterial Morphology
Bacteria are prokaryotic microorganisms characterized by a rigid, polysaccharide-rich
cell wall, a single chromosome unbounded by a nuclear membrane, and no mitochondria or
other membrane-bound organeUes. Individual bacterial cells range in size from -1 to 5 Mm
Bacteria are classified by cell shape (spherical, rod-shaped, filamentous) and arrangement
(single, chains, clumps, pairs, tetrads), by reaction to the Gram stain, and by biochemical
and physiological reactions. The Gram stain divides the bacteria into gram-positive (able to
retain crystal violet stain) and gram-negative (unable to retain crystal violet stain). The cell
walls of gram-negative bacteria contain a lipopolysaccharide called endotoxin. Table 3-2
categorizes some common environmental bacteria according to these characteristics.
TABLE 3 2. MORPHOLOGICAL CHARACTERISTICS OF
SOME COMMON BACTERIA
Genus
Stapkylococcus
Streptococcus
Pseudomonas
Legionella
Bacillus
Mycobacterium
Thermoactinomyces
Mycoplasma.
Gram
Rx
Shape
spherical
spherical
rods
rods
rods
rods
filamentous
spherical,
filamentous
Arrangement
.
clumps
chains
single
single
single, chains
chains
clumps
^-^
3.2.2 Bacterial Physiology
Almost all bacteria require an environmental source of carbohydrate, as opposed to
plants, which can make their own carbohydrates from carbon dioxide (CO2) and water. Most
bacteria are saprophytic (saprophytes), that is, they utilize nonliving organic material.
Bacteria that invade living tissues are parasitic (parasites). The parasitic bacteria are divided
into two groups: (1) facultative, which can utilize both living and nonliving organic material
as food sources and (2) obligate, which must have living tissues to supply growth
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requirements. Saprophytes and facultative parasites can grow in environmental reservoirs,
and can also be grown on culture media.
To transmit infectious disease, bacterial cells must remain viable in air (Kethley, 1957).
Bacterial survival in air is dependent on temperature; relative humidity; presence,
wavelength, and intensity of ultraviolet light; and the -
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populations and their sources, these generalities are useful in interpreting data collected in
indoor environments where complaints have prompted air quality investigations.
3.2.4 Diseases Caused by Airborne Bacteria
Aerosol-transmitted diseases caused by bacteria include infectious disease (e.g.,
legioneUosis), hypersensitivity disease (e.g., hypersensitivity pneumonitis), and toxicoses
(e.g., endotoxicosis) (see Chapter 4).
3.3 FUNGI
3.3.1 Fungal Morpholpgy
Fungi are either unicellular or multicellular. The yeasts are a group of unicellular fungi
that reproduce primarily by budding. Most fungi exist, however, as long chains of cells
called hyphae. Hyphae are often massed into a mycelium. Some mycelia can differentiate
into one or more fruiting bodies (e.g., mushrooms).
Most fungi reproduce by spores that are disseminated through the air. Spores can be
either clones (asexual) of the original plant or can result from genetic recombination (sexual).
Many fungi produce both kinds of spores during a single life cycle. However, most fungal
spores resulting from growth in indoor environments are asexual.
Fungi are classified hi many different ways. The layman description of fungus growth
is usually limited to mold or mildew. More formally, fungi are classified into two groups
based on their mode of sexual reproduction: Zygomycetes (characterized-by a resting
zygospore resulting from nuclear fusion) and the Dikaryomycetes (characterized by a
binucleate multicellular stage preceding nuclear fusion). Most of the fungi that are of
importance in air quality, especially those associated with disease, belong to the
Dikaryomycetes. This large group is further divided into two groups based on the form that
sexual spore production takes: the Ascomycetes, which include most of the common molds,
form sexual spores within a sac; and the Basidiomycetes, which includes the plant rusts,
smuts, and mushrooms, form asymmetrically shaped spores externally on pegs (Burge, 1985,
1989a). '
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3.3.2 Fungal Physiology
Unlike plant cell walls, which are made of a glucose polymer called cellulose, the
fungal cell walls are made of polymers of acetyl glucosamine called chitin. Furthermore,.
fungi are heterotrophic. That is, unlike plants, they do not have chlorophyll and are unable
to synthesize carbohydrates from CO2 and water. Most fungi are saprophytic; however,
some are facultative parasites. A very few of the fungi are obligate parasites, requiring living
tissue to complete their life cycle (Ainsworth and Sussmari, 1965).
As fungi grow, they produce metabolic by-products that, may affect indoor air quality.
The fungi and their by-products have had a major impact on human kind. Antibiotics and
mycotoxins are fungal metabolic by-products. Fungi are also used in the production of some
food items (e.g., cheese, soy sauce) and beverages (e.g., beer, wine) (Kendrick, 1985).
3.3.3 Fungal Ecology
The outdoor air is the primary source for indoor airborne fungus spores. Spores of the
genus Cladosporium usually dominate the air spora during dry weather over most of the
world. During wet weather, ascospores and basidiospores may be predominant (Biirge, 1985,
19§9a; Burge and Solomon, 1990). Agricultural activities provide a primary source for fungi
in outdoor air. Crops that are harvested after the plant dies become well-colonized with
saprophytes that, in turn, become airborne in high numbers during harvesting operations.
The obligatelyjparasitic plant pathogenic fungi are only found in environments where their
host organisms are present, and also may. become airborne when affected plants are handled
(Christensen, 1975):
Water is the single most important factor that determines whether saprophytic fungi will
be found in a given indoor environment. Almost any carbon-containing material can provide
a substrate for fungal growth. However, this growth will not occur if water is not present.
Some carbon-containing materials are hygroscopic and can absorb enough water from the air
(at relative humidities above 60%) to support fungal growth. Condensation on surfaces will
also provide sufficient water. Of course, standing water that contains a carbon source as well
as flooded and/or water-soaked materials will support fungal growth. Any environment
where organic material is stored or handled must be considered contaminated with respect to
both bacteria and fungi. : -
"' - -. 3-7 _ . . . :. -
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Fungal aerosols, as with bacteria, are always mixed with respect to viability so that
viable counts alone always underestimate total counts (Surge et al., 1977a). Factors
controlling airborne survival of fungal spores include availability of water and intensity and*
wavelengths of ultraviolet light (Ingold, 1971). In general, thick-walled colored spores tend
to survive longer than colorless and/or thin-walled spores (Pathak and Pady, 1965).
3.3.4 Diseases Caused by Airborne Fungi
Airborne fungi cause infectious diseases, hypersensitivity diseases, and toxicoses. Some
fungal products may be irritants and contribute to sick building syndrome jsee Chapter 4).
3.4 PROTOZOA
3.4.1 Protozoan Morphology
Protozoa are primarily unicellular organisms that live in water. Many are parasitic and
cause some serious human diseases. Amoebae are protozoans that are amorphous and change
shape by extruding pseudopods. Free-living amoebae are relatively small (8 to 20 /im),
unicellular, eukaryotic organisms that usually contain a single nucleus. They divide by
simple binary fission. Two genera of free-living amoebae have been implicated in indoor
air-related illness: Naegleria and Acanthamoeba. Naegleria is generally slug-like; the
anterior end is broader than the posterior end. Acanthamoeba is characterized by spike-like
cytoplasmic projections. Both move by means of pseudopodia. In addition to its infectious,
trophozoite form, Naegleria can also be transformed into flagellate and ;e^st forms.
Transformation from the trophozoite to the flagellate form usually occursrwhen the supporting
medium is diluted with water. The rapid motility of the flagellate form is by means of two
to four anterior fiagella. When conditions are unfavorable, that is, when food and/or water is
unavailable, oxygen supply is inadequate, or the environment is otherwise unsuitable,
Naegleria can encyst. The spherical cysts are 9 to 12 nm in diameter. A return to the
trophozoite or flagellar state occurs when conditions are once again favorable.
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3.4.2 Protozoan Physiology
The protozoa require environmental carbohydrates and are therefore heterotrophic. The
1 , ' . ' t
carbohydrates can be in the form of dissolved organic material or living or dead cells. Some
protozoa are capable of ingesting gram-negative bacteria, including the Legionella species.
These bacteria may remain alive and virulent within the amoebae, protected from
environmental stresses, including biocides.
3.4.3 Protozoan Ecology "
The free-living protozoa can live wherever water and nutrients are present in sufficient
quantity. .Usually, bacteria are a necessary nutrient source. Protozoa can live at a relatively
wide range of temperatures, .and are found in cold water humidifiers, as well as hot tubs
(Edwards, 1980).
3.4.4 Diseases Caused by Protozoa
Amoebae of the genera Naegleria and Acanthampeba have been implicated in
building-related hypersensitivity disease and possibly infection. If amoebae are present in a
reservoir, that reservoir is contaminated. Given such contamination, there is a potential risk
for sensitization and even infection. The number of amoebae required in a reservoir for
significant risk depends on the dissemination mode.
3.5 ARTHROPODS
3.5.1 Mites
Mites are members of the class Arachnida and the order Acarina. Many different
species of mites are found in homes. The most important species in temperate regions are
Dermatophagoides farinae, D. pteronyssinus, and Eurogfyphus maynei, although other species
may become locally dominant. Mites are not visible in dust, because they are only about
0.3 mm in length. Live house dust mites stay deep inside carpets, furnishings, and bedding.
Originally these mites were often called bed mites. Now it is recognized that very high
levels of mites can also be found in drapes, upholstered .furniture, clothing, and carpets. The
major food source for mites is human skin scales. However, they are also dependent on
. ' . . :' " ' 3-9 '...." '- ' .'
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fungi for growth. Optimal growth requirements for mites are very similar to those
requirements for fungi. The levels of mites and mite'allergens, primarily found in mite
feces, in homes are closely related to humidity (Arlian, 1977). In humid areas, nearly all
homes have mites and up to 90% have greater than the levels now considered to create a risk
for sensitization and asthma (e.g., Florida; Memphis, Tennessee; New Orleans, Louisiana;
southern England; coastal Australia; Sao Paulo, Brazil). Levels of mites in homes in these
areas range from 100 to 18,000 per gram of dust. In drier climates (e.g., Denver, Colorado;
the central part of northern California; inland Australia), levels of mites may be very low,
with 90% of the houses having less than 100 mites/g of dust. Finally, tigre are areas where
the climate is very humid in the summer and then becomes dry in the waiter. This pattern
effects much of the east coast and central United States. In these areas, mites often increase
rapidly during the late summer and decrease steadily over the winter. Detailed studies on
seasonal variations of mites and mite allergens in the United States have been reported from
Cincinnati (Arlian et al., 1982) and Virginia (Platts-Mills et al., 1987). Within each of these
geographic areas, mite prevalence varies between homes for reasons that are as ye^unclear.
3.5.2 Cockroaches
Cockroaches are members of the class Insecta of the order Blattaria. Cockroaches are
present in many homes and can increase to overwhelming numbers if not exterminated
aggressively, and it is now clear that in many inner city areas a significant proportion of
patients with asthma are sensitive to cockroach derived proteins (Bernton and Brown, 1967;
Twarog et al., 1977; Kang et al., 1979; Hulett and Dockhorn, 1979). The German
cockroach (Blatella gemwSca) is probably the most common sensitizerirTthe United States.
Periplaneta americana (the American cockroach), Blaita oriental:*, Periplaneta australisiae,
and Supella supelledium can also become locally abundant and are probably sensitizing. All
of five commercial house dust preparations studied by enzyme-linked immunosorbent assay
(FT ISA) inhibition were found to contain cockroach allergens (Mathews, 1989). In general,
cockroaches are not thought to be vectors of infectious disease.
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3.5.3 Other Arthropods
Many other insects live in houses and can become a source of allergens (e.g., crickets,
house flies, moths, and a variety of beetles) (Mathews, 1989). Occupational exposure data
have provided convincing evidence of the allergenicity of a wide variety of insects including
locusts, crickets, grasshoppers, cockroaches, beetles, moths, blow flies, sewer flies, fruit
flies, and the stinging insects. However, heavy infestation is rare and only occasional reports
of disease association have been made. It is very difficult to prove for each of these cases
that exposure to that;source is contributing to the disease. At present, the only appropriate
measure is to take careful medical .histories and.skin test symptomatic individuals with
extracts of insects that are indicated by the history.
3.6 MAMMALS AND BIRDS
Microorganisms and arthropods are usually uninvited sources of indoor bioaerosols.
However, in many homes, creatures of various sorts are kept as pets. It is estimated that
approximately 100 million domestic animals reside in homes in the United States, the most
common being cats and dogs (Knysak, 1989). Other animals that share the indoor domestic
environment with humans include birds, small mammals (mice, hamsters, guinea pigs), and
snakes. Rodents are also found in laboratory facilities. All of these animals shed proteins
and occasionally bacteria or viruses into the environment. Animal effluents can cause
respiratory allergies and, in rare cases, infectious disease (e.g., lymphocytic choriomeningitis
from virus shed in mouse urine, Q-fever from sheep blood). As many as 30% of allergic
people may be sensitive to domestic animals (Barbee et al., 1981; Fbntana et al., 1963;
Ohman et al., 1977; Ohman, 1978), and 57% of asthmatic children are sensitive to at least
one animal species (Kjellman and Pettersson, 1983). It is estimated that 11 to 30% of those
exposed regularly to laboratory animals experience allergic symptoms (Cockcroft et al.,
1981b; Gross, 1980; Hooketal., 1984; tLutsky and Neuman, 1975). Sources of antigens
include skin scales, saliva, urinary proteins, serum, and feathers. Factors affecting the
abundance of these materials include numbers of animals, time spent indoors, ventilation
rates, and furnishings that can act as reservoirs (see Chapter 4).
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About 25 % of the families in the United States have a cat, and it has been estimated
that 2 mMoncat-aUergic people live with cats despite their symptoms. Possibly 10% of all.
acute asthma in young adults is related to cat allergen exposure. Cat allergens accumulate in
furnishings, and it may take as long as 16 weeks for cat allergen levels to fall after removal
of the cat (Wood etal., 1989). -
In most parts of the world, dogs are of much less importance than cats as a cause of
biological pollution. This probably reflects the fact that most dogs live, at least in part,
outside the house. Acute allergic reactions to dogs are certainly far less common than those
to cats. This'may be because cats are more commonly allowed in bedrooms, exposures are
often more intimate, and cat allergens may be more immunogenic or shed-more copiously
than dog allergens (Knysak, 1989).
Rodents can be present in houses either as domestic pets or as pests, and are commonly
used in the laboratory setting. Laboratory animal allergy has become a severe occupational
problem. Rodents have a common property of leaking protein into their urine. This problem
is particularly well-defined in male rats but is common to all species. These urinary proteins
appear to give rise to sensitization. Individual cases of extreme sensitization to pet rodents
are well recognized as causes of rhinitis or contact urticaria. In addition, rodent urinary
proteins are thought to contribute to asthma, predominantly among children. In areas of this
country where mice and rats are major pests, a significant proportion of allergic patients have
positive skin test to rodent urinary proteins. To date, there have been no epidemiological
surveys to confirm the importance of allergic reactions to rodent urine as a risk factor for
asthma. '
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4. BIO AEROSOL-RELATED DISEASES
4.1 INFECTIOUS DISEASE
More than 100 years have passed since Pasteur demonstrated that airborne
microorganisms can cause infectious disease (Gregory, 1961). Despite the availability of
information from the research of Pasteur and others, emphasis on infectious disease
prevention has focused onrhuman-to^human, direct-contact transmission. This philosophy
assumes that the principles of herd immunity-apply, and relies on immunization and isolation
as the primary means of prevention (Patriarca et al., 1986). Herd immunity assumes that the
number of susceptible people and the nature and frequency of direct, nonaerosol contact
among them determines the rate of spread of infectious disease (Fox et al., 1971). For some
diseases, even though resulting from potentially airborne vectors, these methods are
appropriate. For example, smallpox, a highly virulent, potentially airborne virus, has
ostensibly been eradicated by diligent immunization programs. However, highly virulent
airborne diseases, especially those caused by unstable viruses, are not likely to be controlled
until the dynamics of transmission are understood.
The potential for transmittal of an infectious microorganism via air is dependent on
several factors. First, the disease-causing microorganism must be present in the environment
(reservoir). Second, the microorganism must be able to survive and multiply in that
environment (amplification), and finally,"the microorganisms must become airborne in
sufficient concentration and.remain viable long enough to produce disease (dissemination)
(Feeley, 1985; Surge, 1989b).
For infection to occur, the organism must be virulent and at least one susceptible host
must be present. Virulence is determined by both genetic and environmental factors. Some
microorganisms are inherently more virulent than others. For example, it appears that a
single Mycobacteriwn tuberculosis cell is adequate for infection (Houk, 1980; Riley, 1982),
whereas several hundred Legionella organisms are probably necessary to cause disease
(Meyer, 1983; BaskerviUe et al., 1981). Physiological factors such as life cycle stage are
also important. Some organisms are more virulent during very rapid (log-phase) growth,
whereas other organisms are most infective during the slower, stationary phase.
'.''.''. 4-1
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Environmental factors such as temperature, relative humidity, and radiation can affect both
viability and virulence. Each organism is different with respect to the effects of all of these
virulence factors (Kethley et al., 1957).
The susceptibility of the human host is related to the immune status of that person
(Burge, 1990). Contact with most infectious agents usually results in an immunity to that
organism for a given period of time, possibly for life. For these diseases, immunity can be
induced by immunization or inoculation with parts of the responsible organism. For other
diseases, especially those caused by organisms that change in virulence, immunization is
effective only for short periods. A disease of this latter type is influenza (Selby, 1976).
** »i, , . , ri'-'&v
Factors that damage the immune system will increase, the risk of infection in the
exposed person (Williams et al., 1976; Gardner, 1982). In particular, these factors lower the
thresholds at which pathogens that are otherwise relatively innocuous can cause disease.
Immune system damage can be caused by disease (e.g., fflV infection), immunosuppressive
agents (e.g., cytotoxic chemicals, large doses of steroid hormones) (Hesse et al., 19,86), and
direct damage to cells that function as a part of the immune response within the respiratory
tract (Kark et al., 1982; Petitti and Friedman, 1985; Storch et al., 1979).
4.1.1 Human-Source Infections
The majority of'human-source infections are probably transmitted from person-to-person
by direct contact. Such infections do not stem from a bioaerosol problem and will not.be
considered in this review. However, some very common human-source infectious diseases
are transmitted by air. Logical modes of control of these airborne diseases may lie in the
..pf»
area of indoor air quality (see-Chapter 6). ' ^jU;
, j «'^Sflr
Human-source infections usually rely on the human host to function as a reservoir,
amplifier, and/or disseminator. The virus or bacterium resides in the human,(or animal)
host, is amplified in the host during incubation of the disease, and is disseminated from the
host in respiratory or other secretions^ In general, aerosol-transmitted diseases are respiratory
infections that include coughing and sneezing among their symptoms (Riley, 1982);
however, the act of singing has been identified in the transmission of tuberculosis (Houk,
1980). Airborne human-source diseases rarely occur outdoors because of the large mass of
air available to dilute the aerosol and hostile environmental factors (ultraviolet light,
4-2
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temperature extremes, and humidity extremes). In the indoor setting, rates of infection
depend on the number and virulence of organisms (infectious dose) required to initiate
infection, the number of susceptible hosts in the indoor space, and the number of infectious
doses in the air (Burge, 1990). Low ventilation rates in indoor environments allow for
accumulation of infectious units, often in the presence of an accumulation of susceptible
people.
The human-source infections that are currently considered important with respect to
indoor air quality are influenza, common colds associated with some viruses, measles,
rubella, chicken pox, and tuberculosis. Influenza is an aerosol-transmitted virus disease
(Knight, 1980), although it is still being studied as a direct contact disease by most
epidemiologists (Longini et ah, 1982). It occurs in explosive epidemics, which is
characteristic of airborne transmission, and coughing is a common symptom (allowing
airborne spread). The virus is highly virulent so that only a small dose is necessary for
infection, and the disease is reproduced in volunteers and animals by aerosol more easily than
by nasal instillation (Knight, 1980; Schulman, 1968). A contained epidemic aboard a
commercial airliner was unequivocally caused by air transmission resulting from a single
active case of influenza and a period "of inadequate ventilation (Moser et al., 1979).
At least 100,000 episodes and 13,000 excess deaths are attributable to influenza each
year (Garibaldi, 1985; Schoenbaum, 1987). Although influenza-related mortality is highest
in the elderly, morbidity is~ greatest in children. Some evidence exists for the hypothesis that
influenza epidemics begin in the schools (Monto, 1987). Immunization programs for the
elderly population .may decrease mortality, but will not halt the spread of this serious and
costly disease. Dilution ventilation in the school room is a control approach that deserves
attention, but is not popular because it conflicts with energy conservation policies.
There is still controversy over the mode of transmission (direct contact vs. aerosol) of
the common cold viruses. However, Couch (1981) was able to experimentally transmit the
coxsackie virus between volunteers in a situation where direct contact was prevented. Similar
disagreement exists for the rhinoviruses, with some investigators presenting data for or
assuming direct contact (Longini et al., 1988) and others for the aerosol route of infection
(Dick et al., 1987). Colds are caused by many different viruses, some of which do not
survive well in aerosols, and are probably transmitted primarily by direct contact in most
4-3
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situations. Under conditions that favor aerosol survival, however, airborne transmission can
occur. Some human-source infections, such as influenza, are most readily infective by lower
airway challenge, rather than nasal instillation, and are probably primarily airborne (Knight,
1980).
At least 90,000,000 episodes of the common cold occur each year in the United States,
resulting in 200,000,000 days of restricted activity (Dixon, 1985). Attempts'* control this
common disease have relied on interruption of direct-contact transmission (biocidal
handkerchiefs) or immunization. Neither of these methods have been effective. Brundage
et al. (1988) has demonstrated that inadequate ventilation may facilitate thread of
adenbvirus in army recruits. His study was not properly controlled, however, and should be
repeated.
Measles, rubella, and chicken pox, the common childhood diseases, are all aerosol-
transmitted viral diseases (Habel, 1945). The measles virus is so virulent that only
4 infectious units/minute released from an infected host can initiate an epidemic (Riley,,
1980). Resistance to measles requires either previous infection or vaccination. Because of its.
virulence, measles usually infects all exposed susceptible hosts. Therefore, those remaining
sensitive are mostly very young children and a few people bom before active immunization
programs were instituted (Davis et al., 1986). Measles virus has been documented to travel
through ventilation system components to infect distant susceptible people (Bloch et al.,
1985). In such a situation, all susceptible people usually become infected. Immunization
alone is unlikely to control this disease. Potentially more effective measures have been
proposed including: . ...
. disinfecting air in high-risk enclosed spaces (such as schools) with ultraviolet light
(Riley, 1980),
. more effective control of recirculation patterns in clinical spaces (Davis et al., 1986),
_and
. increasing fresh air ventilation rates (i.e., increasing percentages of outdoor air in
recirculation systems).
4-4
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Control by environmental intervention is only effective if the environment being treated is the
only transmission site. It is not useful to treat air in a school if the disease is being
transmitted on school buses. Careful analysis of suspected sources is essential to effectively
control all environmentally transmitted diseases (Wells et ah, 1942).
Rubella, a mild disease in children, presents a significant risk of birth defects when
contracted by pregnant women. Most attempts at control of rubella have centered on
immunization of school-age children based on the assumption that they spread the disease at
school and bring it home to their pregnant mothers. It appears, however, that women are just
as likely to get me disease.directly from public contact in poorly ventilated spaces (e.g.,
public transportation) (Langmuir, 1980).
Chicken pox is probably the most contagious of infectious diseases and is transmitted
by air whenever an infected person coughs (Couch, 1981). Epidemics have been recorded in
hospitals (Gustafson et al., 1982; I
-------
4.1.2 Environmental-Source Infections
Environmental-source infections result from exposure to reservoirs where saprophytic
organisms are amplified and/or nonhuman (usually mammalian), living reservoirs. Any
environment containing some kind of an organic carbon source, available nitrogen, and water
can be home to one or more saprophytic organisms. Fortunately, most of these organisms
cannot invade human tissue and do not cause infectious disease. Very few primarily
saprophytic organisms can invade a normal healthy human who possesses an intact immune
system. However, a few saprophytes (usually those that are- adapted for growth in
environments in which temperatures are maintained in the range of human .body temperature)
'.^i/?
will attack minimaUy compromised individuals (e.g., those who are heavy,smokers), and
many will cause disease in severely compromised hosts (e.g., AIDS patients and patients who
are on immunosuppressive medication to prevent transplant rejection). The environmental
infectious agents can be divided into two general categories:
(1) the primary fungal pathogens (including Histoplasma, Coccidioides, and
Blastomyces), and
(2) the opportunistic pathogens (including Legionelld, and many organisms that are
facultative parasites). ' ' .
The primary fungal pathogens grow and reproduce in nature as soil saprophytes,
producing mycelium and spores as ordinary fungi do. Hov/ever, when spores of these fungi
gain access to the human respiratory tract, they are able to adapt and growjn this unusual
environment/and produce-disease. Histoplasmosis, cryptococcosis, cocadipidornycosis,
blastomycosis, and sometimes sporotrichpsis are fungal diseases of this sort. In normal
people, these diseases are usually self-limiting. However, when immune system defects are
present, the diseases can be serious or. fatal. Although these diseases all have primary foci
that are outdoors, the outdoor aerosol can penetrate into interiors, and, especially where
debilitated people reside, they can present significant problems.
Histoplasma is very common in the Americas, and resides in soil enriched with bird
droppings. Primary focus of exposure is outdoor air during disturbance of contaminated soil.
Indoor air exposure may occur when such soil is disturbed adjacent to open windows or air-
4-6 ' .
-------
intakes. In most cases, the disease, histoplasmosis, is subclinical (does not produce
noticeable symptoms) and self-limiting. However, it can be severe or fatal in
immunocompromised individuals. It is estimated that 40,000,000 people in the United States
alone have had histoplasmosis, and that there are 200,000 new infections each year (Ajello,
1971; Furcolow, 1958).
Cryptococcus neoformans is almost exclusively associated with pigeon droppings and
may be the predominant organism in old, dry droppings in roosting areas (Emmons, 1955).
It does not compete well with other organisms and is rapidly overcome when soil is mixed
with infected debris. .Indoor exposure may occur when old pigeon roosting areas in attics are
disturbed. As with histoplasmosis, the disease is usually subclinical and self-limiting in
normal people, but can become severe and fatal in immunocompromised patients. Because a
sensitive antibody assay is as yet unavailable, accurate estimates of the incidence of this
disease are also not available. One estimate suggests mat 15,000 cases occur each year in
New York City alone (Kaufman and Blumer, 1978).
1 Coceidioides grows in dry soils (hi the semi-arid southwest United States and Mexico)
> ' , ... * . " , »
with high concentrations of carbonized organic material and high salt concentrations. Spores
become airborne when contaminated soil is disturbed. Epidemics often occur during
\ " t " , -
sandstorms. Most exposure occurs outdoors, but spores may enter the indoor environment.
Coceidioides may be the most virulent of the fungal pathogens. A few spores are sufficient
to cause disease in a host with a normal immune system, whereas a massive exposure will
cause serious disease (Larsen et al., 1985). Risk factors for the development of serious or
fatal coccidioidomycosis have yet to be clearly established, although an immune defect is
suspected (Kirkland and Fierer, .1985). Probably more than 100,000 cases of
coccidioidomycosis occur per year in the United States, most of them concentrated in the arid
southwest.
Blastomyces is endemic in the eastern United States. The organism inhabits wet soil
' enriched with animal manure, cannot withstand drying, and does not compete well with other
soil microorganisms (Klein et al., 1986). Epidemics of the disease (blastomycosis) are
usually associated with soil disturbance, including construction activities (Kitchen et al.,
1977); Whether or not blastomycosis exists as a subclinical disease is not known. The
'4-7
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disease does not produce lung or serological changes in recovered individuals, and no
accurate test is available to assess rates of occurrence.
The opportunistic pathogens are saprophytes that normally occupy natural environments,
but cause infectious disease when they penetrate susceptible human hosts! In these cases,
susceptibility implies some lowering of defenses rather than absence of specific protective
antibodies. Any factor (i.e., disease, smoking, alcohol or drug abuse, chemotherapy) that
damages the human immune system can render a person more susceptible to these disease
agents. In some sense, the primary fungal pathogens are opportunistic in that they are serious
diseases only in those with some immune dysfunction. The true opportunists, however, do
J . , -*ia.*i-
'not apparently cause disease at all in people with normal immune systems. Some common
opportunistic pathogens that cause airborne disease are the bacteria Legionella pneumophila,
Pseudomonas, and Acinetobacter, many fungi, especially those able to grow at elevated
temperatures; and a few protozoa.
Legionella is the most notorious of the opportunistic pathogens and has been extensively
reviewed (Meyer, 1983; Winn, 1985; Davis and Winn, 1987). Legionella is a common
environmental saprophyte, and it has been isolated from soil, water, and other outdoor
environmental reservoirs. In addition, it can contaminate air conditioning equipment, potable
water, humidifiers/nebulizers and other respiratory therapy equipment, whirlpools/spas,
sprinkler systems, and industrial coolants (Winn. 1985; Davis and Winn, 1987; Doebbeling
and Wenzel, 1987; Burge, 1990). It causes two distinct clinical syndromes: a bacterial
pneumonia that carries a low attack rate but high mortality (Legionnaires' disease) (Eraser
et al., 1977), and a nonpneumonic disease with a high attack rate and rapid recovery (Pontiac
fevert (Glick et al., 1978). Most Leg/one/Za-reiated epidemics have beentraced to Legionella
, *v3Jj-AiVri.
pneumophila serotype I, although other serotypes and other species have" been implicated in
isolated cases and unusual epidemics (Plouffe et al., 1983). Airborne transmission has been
clearly demonstrated (Baskerville et al., 1981; Davis et al., 1982). Immune suppression is a
risk factor for Legionnaires' disease, especially suppression of lung defenses. As with all
opportunistic diseases, normal healthy people are rarely at risk, whereas patients with severe
immunosuppressive disease and those on immunosuppressive medication become infected with
apparently low dose exposure (Davis and Winn, 1987; Guiguet et al., 1987). In addition,
cigarette smoking and excess alcohol consumption appear to be risk factors (Storch et al.,
4-8
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1979). Legionnaires' disease is not rare. At least 4% of the American population has
anti-Legioneffa antibodies (Winn, 1985) and more than 20,000 community-acquired cases
probably occur each year, with an additional 200,000 acquired in hospitals (Meyer, 1983).
Bacteria other than Legionetta have been shown to cause pneumonia in high-risk
populations. Pseudomonas and Acinetobacter may inhabit respiratory therapy equipment in .
medical facilities and humidifiers in home and work environments (Grieble et al., 1970;
Smith and Massanari, 1977; Kelsen and McGuckin, 1980; Spendlove and Fannin, 1983;
Williams et al., 1976). Incidence of community-acquired disease related to these types of
exposures are unknown. Nosocomial infections from these types of sources are probably
relatively common.
Control ofLegionella and other bacterial saprophytes depends on preventing
accumulation of stagnant water in the indoor environment, preventing entrainment of cooling
tower effluent into the indoor space, and maintaining adequate temperature and/or
chlorination of hot water systems, especially in hospitals. Potential health effects from the
use of these preventive measures, (e.g., effects from exposure to biocides, risk of scalding
from hot water) must be compared to the risks associated with bioaerosol exposure (Stanwick,
1986). It should be noted that the risk of opportunistic infections are low for normal people.
The best known opportunistic fungal pathogen is Aspergillusfiamgants. The organism
produces toxicoses and allergies, grows in mucus secretions in the human respiratory tract,
and can invade living tissue (Rippon, 1988). It is an ubiquitous fungus that occupies natural
and man-made environments where significant heating occurs (30 to 45 °C) (Emmons, 1962).
Although A. fumigatusis. the most common fungal agent in cases of infectious disease,
Rinaldi (1983) lists. 22 species that have been implicated in human infectious ,
disease (Table 4-1). According to Rippon et al. (1965) many Aspergitius and Penicilliwn
species can become pathogenic. Pathogenesis appears to be related to temperature tolerance.
Solomon et al. (1978) recovered 10 species of thermotolerant aspergilli from air in a
midwestem hospital in addition to representatives of 10 other thermotolerant genera.
Human infection with the opportunistic fungi depends on immune dysfunction, and
diagnosis of invasive aspergillosis or fungosis should be considered indicative of underlying
disease. The number of spores required to initiate infection is unknown. Probably one
viable spore is sufficient in a severely compromised host, whereas a healthy normal person
4-9
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TABLE 4-1. ASPERGJLLUS SPECIES IMPLICATED IN
CASES OF INFECTIOUS DISEASE
A. amstelodami _ , A- nidulans
A. candidus A' Ser
A. carneus A- *****
A. conicus A. ochraceus
A. deflecms A' °W*
A.fischeri A. parasincus
A. flavipes A-
A. flams A-
A. jumigants A- terreus
A. famigants var elttpticus A-
A. glaucus group _ A- ^rsicolor
Source: Rinaldi, 1983; Rippon, 1988
can resist infection when exposed to millions of spores. Epidemics of aspergillosis in
hospitals have been traced to environmental contamination of fire-proofing materials (Aisner
et al., 1976), renovation activities (Arnow et al., 1978; Krasinski et al., 1985), road
construction, and contaminated air conditioners (Lentino et al., 1982). The risks of disease
resulting from the constant background of Aspergillus in air is not known (Solomon et al.,
1978). The disease is of major concern in transplant facilities, for AIDS patients, and for
patients on high-dose steroid "therapy. Although Aspergillus is the mostgDtorious, many
other fungi can become invasive pathogens under similar circumstances. Candida is not
known to be commonly airborne, but it leads the list of opportunistic fungal pathogens,
followed by Aspergillus, Crypwcoccus, and the zygomycetes (e.g., Mucor, Rhizopus), all of
which are commonly transmitted by air. The incidence of opportunistic fungal infection is
not known. The disease is often fatal. Control depends primarily on prevention of exposure.
Unfortunately, Aspergillus and other fungi reside in the human nose, and can probably cause
infection when inhaled into the lower airways from this source (Walsh and Pizzo, 1988).
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4.1.3 Animal-Source Infections
Under some circumstances, some microorganisms that regularly inhabit other animal
species can infect humans via the airborne route of exposure (Spendlove and Fannin, 1983).
The best known of these are Q-fever, anthrax, and brucellosis; however, the incidences of
these diseases are probably quite low.
Q-fever is a rickettsial disease that is endemic in sheep. Epidemics of this disease have
occurred in medical facilities where sheep were being used for research (Bayer, 1982;
Meiklejohn et al., 1981; Huebner, 1947; Schachter et al., 1971) and in meat-handling plants
(FeldmanetaL, 1950; Sigeletal., 1950); '...'
Anthrax is also known as wool sorters disease because the endospores produced by
Bacillus anthracis (the causative agent) can survive for long periods of time on the wool of
infected animals (Dahlgren et al., I960; Young et al., 1970). Fortunately, the infective dose
of this organism appears quite high (> 1,300 units). ' .
Brucellosis is generally a disease of domestic animals; however, it can be contracted by
people in the meat-packing industry (Buchanan et al., 1974; Hendricks et al., 1962;
Huddleson and Munger, 1940; Kaufmann et al., 1980). A less well-known animal disease is
lymphocytic choriomeningitis, a viral disease that occurs in rodents. The causative agent can
be isolated from rodent urine and may become airborne if the urine is disturbed. Epidemics '
have occurred in animal vivaria and sporadic cases in homes may have resulted from
exposure to urine from infected house mice (Couch, 1981).
4.2 HYPERSENSmVTTY DISEASE
The hypersensitivity diseases are caused by individual immunologic sensitization to
specific antigens, substances that can trigger an allergic response. Antigens or immunogens
are able to stimulate production of antibody or antigen reactive cells and serve as specific
targets for the antibody or sensitized cell produced. Proteins, lipoproteihs, glycoproteins,
polysaccharides, lipopolysaccharides, larger polypeptides, and nucleic acids are all potential
antigens. Most antigens are 5,000 to 50,000 daltons in molecular weight and are soluble, a
necessary requirement for antigenicity. A number of smaller, highly reactive molecules may
/also be antigenic, provided they are able to bind to larger carrier molecules. These small
' . ' " ' ' ' . ; '' .4-11 '" ' .. ' .'"... "
-------
molecules are known as haptens. Common haptens include metal salts, isonicotinic acid
hydraride, trimellitic anhydride, and other highly reactive chemicals. Haptens usually cause
sensitization in occupational settings where exposure occurs over long periods of time. An
important characteristic of antigens, with the possible exception of some haptens, is that they
do not elicit toxic effects in the absence of an immune response. Thus, nonallergic
individuals have no significant symptoms from exposure to dust, even in homes with high
levels of dust mite, cockroach, cat, or fungal proteins.
Because antigens produce immunological changes in allergic individuals, it is possible to
identify sensitization to specific antigens. Once an individual becomes ^sensitized to a
particular antigen, subsequent exposure produces an allergic response. ..Forms of sensitization
of concern for indoor air are specific immune responses involving either antibodies or .
T cells. Determining sensitization provides two kinds of information. First, if. is an
indication that the individual has been exposed to an antigen. Second, the form of
sensitization may act as a guide to the immunopathology of the associated disease.
There are three forms of immunity that have to be considered hi the discussion of
indoor biological pollution: (1) IgE antibody response, (2) IgG antibody response, and
, (3) T cell response. The IgE antibodies produce immediate hypersensitivity when exposed to
an immunogen/" These antibodies can be detected by wheal and flare skin responses or by
serum assays. IgG antibodies. are an important part of protective immunity and are also
associated with some forms of hypersensitivity. IgG responses to antigen are only detected
through serum immunoassays. T cell responses produce delayed hypersensitivity and in
clinical practice are detected by 24 or 48 hour indurated erythematous skin responses.
The hypersensitivity -diseases most clearly associated with mdoor-ajriguality are asthma
and allergic rhinitis and hypersensitivity pneumonitis. Asthma and rhinitis are associated with
IgE antibody responses possibly, for some antigens; IgG response; and a specific form of.
T cell response. Hypersensitivity pneumonitis is also an IgG antibody and a T cell response.
However, the form of T cell response differs from that of asthma and rhinitis.
4.2.1 Rhinitis, Asthma, and Allergic Bronchopulmonary Aspergillosis
Allergic rhinitis, allergic asthma, and allergic bronchopulmonary aspergillosis (ABPA)
result from interactions between antigens or allergens and IgE antibodies. They affect peopfe
4-12
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who have the genetic tendency to develop IgE antibodies usually in response to inhaled or
ingested allergens.
Allergic rhinitis (hay fever) affects up to 20% of the population and, in some instances,
exposure to the appropriate allergen may have an incapacitating effect. Allergic rhinitis is
characterized by nasal itching, congestion, runny nose, sneezing, and watery eyes.
An estimated 10 million people in the United States have asthma. In 1987,
4,360 people died from asthma compared to 2,891 people in 1980 (U.S. Department of
Health and Human Services, 1991). Asthma is characterized by chest tightness, wheezing,
' *
cough, and shortness of breath. Symptoms may'occur within an hour of exposure to the
allergens or may be delayed in onset for 4 to 12 hours. For many years it was assumed that
airborne allergens caused asthma entirely by the release of histamine from mast cells in the
lung. This belief was held because inhaling allergen extracts produced a rapid asthmatic
response similar to, that produced by histamine. However, closer observation of asthmatic
individuals revealed that provocation with allergen often produced a delayed or late airway
response as well as the immediate response (Booij-Noord et al., 1972; Warner et al., 1978).
This late response is not seen with histamine and is associated with increased bronchial
irritability, which can last for days or weeks (Cartier et)al., 1982). It is now believed that
this late bronchial response is caused by inflammatory cells other than histamine, in addition
to possible epithelial damage in the airway (U.S. Department of Health and Human Services,
1991). In keeping with this, it has been shown that prolonged avoidance of exposure to
house dust can lead to marked reductions in nonspecific bronchial reactivity (Kerrebijn, 1970;
Platts-Mills et al., 1982; Charpin et al., 1988). Chronic exposure to low levels of indoor
allergens over days or weeks might be more important than a'larger amount over a short "
period. ,
The size of antigen-containing particles does not appear to be critical for development of
IgE-mediated disease. Approximately 5% of particles as large as 15 or 20 pm will enter the
bronchial tree (Task Group on Lung Dynamics, 1966; Svartengren et al., 1987).
Allergic bronchopulmonary aspergillosis is a disease of asthmatics where a fungus (often
Aspergillus) colonizes the mucus secretions in the lung. Symptoms are similar to pneumonia.
Patients develop both IgE and IgG antibodies against the specific colonizing fungus. The
' 4-13
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disease is apparently related to host factors rather than intensity of exposure to viable spores
(Slavin, 1983).
4.2.1.1 Causative Agents
Biological agents known to produce antigens that cause allergic rhinitis and asthma
include fungi, algae, pollen, plant parts used as food, arthropods, and avian and mammalian
effluents. Probably any protein or glycoprotein derived from any living organism can be
allergenic if a predisposed person is appropriately exposed. Some antigens that are
considered of primary importance epidemiologically in indoor air have been identified and
^£C^WiJ*ir
characterized. However, with respect to the different .kinds of possible antigens in the indoor
environments, most remain unknown.
It is estimated that 30 to 45% of attacks of acute asthma in children over 7-years old
and in adults under 50 years of age can be attributed to indoor allergen exposure (Pollart
et al., 1989a; Gelber et al., 1990). Of the 2 .million estimated annual emergency room visits
for asthma, as many as 400,000 of these cases can be attributed to exposure to indoor
allergens.
For sensitizing agents (allergens) there are two separate groups of individuals potentially
at risk:
(1) those individuals who are exposed to a level of antigen that is considered sufficient
to give rise to sensitization, and
(2) those individuals who are sensitized and continue to be exposed and develop
v ' ' .-l_i *
symptoms. v --.^-r .
For some of the major sources of indoor biological pollution, reducing exposure is not
simple and, therefore, it has been difficult to obtain clear results showing that avoidance
improves the disease (Korsgaard, 1982; Burr et al., 1980). There are, however, at least three
studies showing improvement in asthma in mite-allergic individuals when exposure was
decreased (Murray and Ferguson, 1983; Mitchell et al., 1985; Walshaw and Evans, 1986).
For most allergens, little is known about -the actual dose-response relationship. To
i t
determine the dose-response relationship, the allergen must first be identified and standard -
4-14
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assays must be developed. Epidemiological surveys, including field measurements and
assessment of disease rates, may also assist in defining the dose-response relationship. It
should be noted, however, that any fixed threshold is simply a statistical value for exposure
above which a given proportion of the population would be expected to develop sensitization
and, if exposure continues, a proportion of the sensitized individuals will develop symptoms.
All fungi that have been used in skin-testing surveys have been shown to elicit reactions
in some fraction of the allergic (IgE producing) population. This probably means that all
fungi are potentially antigenic, and patterns of exposure may control the importance of each
in allergic disease (Burge, 1985). Research on the biochemical characteristics of antigenic
material derived from the fungi is limited. Semipurified antigens have been produced from
AUernaria alternata (Yungihger et al., 1980), Cladosporium herbarum (Aukrust and Borch,
/ ' ~. ' '"
1979), and Aspergillus fimigatus (Kim and Chaparas, 1978). Other fungi have been
extracted and the antigenic materials have been concentrated (Homer et al., 1988; Horner
et al., 1989; Davis et al., 1988). Some antigenic cross-reactivity may exist between different
fungi (Agarwal et al., 1982; O'Neil et al., 1988). However, in many cases, batch and strain
variability has not been considered. The overwhelming numbers of fungi to which people are
commonly exposed in the indoor environment, the intrinsic variability of fungi in culture, and
the fungal enzymes always present during extraction procedures make the task of
identification of even the major allergens formidable (Surge, 1989a).
Several of the important allergens of dust mites have been purified, cloned, and
sequenced (Platts-Mills and Chapman, 1987; Chua et al., 1988). Some mite allergens are
predominantly found in mite feces and are now known to be digestive enzymes.- Structurally,
the main allergens are -glycoproteins with a molecular weight of 15,000 to 30,000
(Platts-Mills and Chapman, 1987; Yasueda et al., 1989). Another important characteristic is
that they are rapidly soluble in salt water.
There are multiple studies on the relationship between exposure to the dust mite and
sensitization (Korsgaard, 1983; Peat et al., 1989; Sporik et al., 1990; Platts-MUls et al.,
1986a; Wood et al., 1989). Based on this information, the following provisional standards
have been proposed for exposure/allergic response by an international workshop:
4-15
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2 fig Group I allergen/g of dust represents a risk for development of sensitization and
bronchial reactivity, and
« 10 /tg Group I allergen/g of dust present a risk for the development of acute asthma
(Platts-Mills and de Week, 1989).
From studies on large numbers of samples, it has been concluded that 100 mites/g of
dust is equivalent to 2 /tg of Group I allergen. It has been suggested that if all houses in an
area contain >2 pg Group I mite allergen/g of dust, then the susceptible individuals, perhaps
as many as 30% of the population, will become sensitized. It is estimated that 10% of the
population of the United States is sensitized to mite antigen. .^^
The occurrence of respiratory symptoms from the inhalation of other insect-derived
material has been well documented for more than a half century and may be a more prevalent
problem than is currently appreciated. Much of the data on insect allergies is occupational.
Locusts, crickets, grasshoppers, and cockroaches are grown for a variety of purposes (usually
laboratory) and have induced sensitization in workers. Beetles, moths, butterflies, and flies
have also been implicated in occupational allergies resulting from inhalation (Mathews,
1989).
Domestically, cockroaches are probably the most potent insect source for airborne
allergens. Occupants of the poorer sections of large cities are more at risk of this kind of
exposure, and cockroach antigen probably replaces mite antigen as the most important inducer
of childhood asthma in this environment (Bemton and Brown, 1967; Mendoza and Snyder,
1970; Schulaner, 1970). The German cockroach (Blatella gerrrwnicd) appears to be the most
prominent sensitizer m'ffierUaated States, but others may produce immujwripgic responses as
well. Several different proteins between 70,000 and 75,000 daltons have been identified as
important cockroach allergens (Stankus and O'Neil, 1988). In contrast to mites, cockroach
feces is apparently not the primary source of antigenic material. Cast skins and whole body
extracts appear to be more potent sources (Richman et al., 1984); however, levels of antigen
that induce sensitization or produce symptoms remain unknown.
Other insects that cause sensitization in homes are the cat flea (Rolfsen et al., 1987),
mushroom flies (Truitt, 1951), silk worm secretion (silk) (Dewair et al., 1985), and many
others (Mathews, 1989). People who maintain fish hi tanks may develop sensitivity to
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chironomids kept as fish food (Baur et al., 1982). In the lease of chironomid sensitization,
10 different hemoglobins have been found to be allergenic. It may be that, as is the case for
fungi, any insect to which people "are appropriately exposed over a long period of time can
induce sensitization. ,
Allergic reactions to animal-derived allergens (danders) are frequent and have been
known for many years. The allergic reactions result from exposure to domestic animals or to
laboratory animals, especially rodents. In Sweden, more than 5% of unselected children
. * »
were found to be sensitive to animal allergens (Kjellman and Pettersson, 1983). As many as
30% of individuals frequently .exposed to laboratory animals in vivaria experience symptoms
(Cockcroftet al., 1981b; Gross,, 1980; Lutsky and Neuman, 1975).
In 1971, the first purification of a cat allergen established that this protein was present
in saliva. The protein, which is now designated Felis domesticus 1 (Pel d I), is a
37,000 dalton, freely soluble glycopfotein, and has been identifiable for 15 years by a
conventional antiserum (Leitermann and Ohman, 1984; Ohman et al., 1987; Chapman et al.,
1987). Possibly 10% of all acute asthma in young adults is related to cat allergen exposure
of patients who have IgE antibodies for cat proteins (Pollart et al., 1989a; Gelber et al.,
1990). It has been suggested that a threshold level of 8 fig cat allergen/g of dust may be
sufficient to produce sensitization (Gelber et al., 1990). Approximately 2 million Americans
who are allergic to cats live in a house with a cat. '
Canis familliaris I (Can F I) has been proposed as the major dog allergen (Schou and
Lowenstein, 1990; de Groot et al., 1990). Urine, serum, and saliva (which contains serum
proteins) all contain potent allergens (Viander et al., 1983). Although breed-specific
allergens have been hypothesized,"it appears that variation'in reactivity to different canine
breeds results from concentration differences between shared allergens (Knysak, 1989).
Urine and possibly saliva are the primary allergen sources for rodents. Two major
allergens have been purified from rat urine; one primarily confined to male rats (Longbottom,
1980). .Two major mouse allergens have been isolated; one from urine (Lorusso et al., 1986)
and one from dander (Price and Longbottom, 1987). Three antigens have been recovered
from guinea pigs; two from urine and one from other sources (Walls and Longbottom, 1983;
Walls et al., 1985). As with the fungi and insects, it appears that any mammalian effluent
can act as an allergen.
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4.2.1.2 Diagnosis of Immediate Hyper-sensitivity
Patients with allergic diseases, such as hay fever, perennial rhinitis, anaphylaxis, and
many cases of asthma, produce sk responses with a wheal (or hive) and surrounding flare
(redness) when an extract of the causative allergen is introduced into their skin. The reaction
occurs usually within 10 minutes, hence the term "immediate hypersensitivity". Evaluation
of the significance of the skin test response depends on
the quality and strength of the extract,
the technique of skin testing, and .^
" ',, ' ;, . */(iV'
the criteria used for identifying a positive respoase." ,^,
There are well-established techniques for preparing and storing extracts and evaluating
their strength hi vivo and in vitro (Aas et al., 1978; Dreborg and Enarsson, 1990).
Recently, monoclonal antibodies have allowed much simpler purification and assay, of Pel d I,
a cat allergen (Chapman et al., 1988). However, until specific allergens are characterized
and assays are developed to accurately measure them in extracts, allergen standardization will
remain a significant problem.
Two general types of skin tests are commonly used: the epicutaneous (scratch, prick)
test and the intradermal test. Skin testing for immediate hypersensitivity carries the risk that
patients may rapidly develop generalized hives, hypotension, and/or airway obstruction (i.e.,
anaphylaxis). However, the risk of fatal anaphylactic reactions following epicutaneous or
prick tests is so low that it can reasonably be ignored. By contrast, intradermal tests,
particularly if carried out: without a screening prick test, carry a significant risk of fatality
'.f^Sis-^
(though even this is less than 1:2,000,000) (Lockey et al., 1987). Thus, skin testing should
only be carried out by individuals familiar with the test and in the presence of a physician.
Resuscitation equipment should also be readily available. Intradermal testing should only be
considered after negative epicutaneous tests.
The primary objective of skin testing is to identify whether the individual being tested
has made an immune response. Criteria for reliability of skin testing include repeatability
and correlation with other methods of testing (i,e., challenge tests or serum assays)
(Platts-Mffls et al., 1982).
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For some of the indoor inhaled allergens, the relationship to a disease is obvious to the
exposed individuals. This is particularly so for cat allergens where the: onset of rhinitis,
asthma, or conjunctivitis may follow within 5 to 15 minutes of entering a house with a cat.
For other allergens, the relationship is less obvious. For these allergens, formally
establishing the relationship can be achieved with challenge studies to determine whether
exposure to a specific allergen will produce rhinitis or asthma. Epidemiological studies on
random populations may assist in showing that sensitivity or exposure to a specific allergen is
common among individuals with the disease. Documented cases showing an improvement or
subsidence of symptoms once subjects have been removed from exposure may also strongly
support the relationship of exposure and disease (Platts-Mffls etal., 1982; Kerrebijn, 1970;
Charpin et al., 1988).
4.2.2 Hypersensitivity Pneumonitis
Hypersensitivity pneumonMs (HP), also called allergic alveolitis, reflects both .
antibody-dependent mechanisms and cellular immune responses (cell mediated immunity). It
is characterized by recurrent pneumonia with fever, cough, chest tightness, and lung
infiltrates. Progressive, irreversible lung damage may occur with continued exposure to
antigens. High levels of IgG antibodies directed against the causal antigen are produced and
/can be used as a diagnostic tool in attempting to make connections between the environmental
exposure and a specific disease (Fink, 1983).
4.2.2.1 Causative Agents
Any organic dust capable of penetrating the lower airways and present in high
concentrations can probably cause HP (Salvaggio, 1987; Pepys, 1969). In addition, many of
the agents known to be important in HP are, in themselves, adjuvants (agents capable of
stimulating the immune system). For example, the thermophiUc actinomycetes, which'are
common causes of HP epidemics, have been shown to have adjuvant activity (Bice et al.,
1977). Most antigen exposures are mixed with respect to organisms. It may be -that
exposure to, for example, fungal spores, is only likely to cause HP in the presence of a
known adjuvant, such as endotoxin. Biological agents that produce antigens known to cause
HP include bacteria, fungi, protozoa, birds, and mammals. In fact, all-the biological agents,
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with the exception of the arthropods and plant pollen, that have been shown to produce
antigens stimulating IgE-mediated dise. -, have also been implicated in HP,
The classic form of HP is called farmer's lung disease, and is caused by inhalation of
the minute spores produced by the thermophilic actinomycetes (e.g., Micropolysporafaeni,
Itemwacnnomyces vulgaris) (Kobayashi et al., 1963; UBerge and Slahmann, 1966; Gregory
et al., 1963; Gregory and Lacey, 1963; Lacey and Lacey, 1964). These organisms are
abundant in organic material during degradation by other microorganisms (other bacteria,
fungi). Bagassosis (HP resulting from exposure to moldy sugarcane bagasse) (Buechner
et al., 1958; Salvaggio et al., 1966) and mushroom worker's lung (HP |om exposure to
moldi composting material) (Bringhurst et al., 1959) are also mediated b^the thermophilic
actinomycetes. Thermophilic actinomycetes have also caused HP when present in water-
spray cooling systems (Banaszak et al., 1970), water in ventilation ductwork (Hales and
Rubin, 1979), home humidifiers~(Fink et al., 1971; Sweet et al., 1971; Burke et al., 1977),
car air conditioners (Kumar et al., 1981), dust in air ducts (Weiss and Soleymani, 1971),
console humidifiers (Tourville et al., 1972), and evaporative air coolers (Marinkovich and
Hill, 1975). Glycopeptide and protein antigens have been purified from the thermophilic
actinomycetes, although purification and complete characterization has not been achieved
(LaBerge and Stahmann, 1966; Pepys and Jenkins, 1965; Wenzel and Emanuel, 1965;
Edwards, 1972; Fletcher et al., 1970).
Other kinds of bacteria have also been suspected to cause HP. Humidifiers
contaminated with Flavobacterium (Rylander et al., 1978), Bacillus sereus (Kohler et al.,
1976), and Bacillus subtilis (Parrott and Blyth, 1980) have been implicatedjn HP epidemics.
Bacillus subtilis was also suspected as the causative agent in wood dusl^(Johnson et al.,
1980).
Suberosis, Sequoiosis, and cheese worker's lung are forms of occupational HP outside
the farmini environment. Penidllium species have been involved in a number of HP cases
and epidemics (Bernstein et al., 1983; Campbell et al., 1983; Fergusson et al., 1984; Solley
and Hyatt 1980; van Assendelft et al., 1985). Aspergillus species have been less frequently
implicated in the development of HP (Blyth, 1978; Patterson et a!., 1974; Vincken and
Roels, 1984; Yocum et al., 1976). Other fungi that have been reported to cause HP include
Crypwstroma conicale (Emanuel et al., 1966), Phoma violacea (Green, 1972), Merulius
4-20
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lacrymans (O'Brien et ail., 1978), Cephalosporiwn (Patterson et al.t 1981), Alternaria
(Schlueter et al., 1972), and Trichosporon citianewn (Shimazu et al,, 1984). Most of these
fungi have spores that are less than 5 >m in aerodynamic diameter.
In addition to other diseases, the free-living amoebae can produce HP by excreting
antigens into water. In one report, Naegleria was implicated'as the causative agent in a case
of humidifier fever (Edwards et al., 1976; Baxter, 1982; Cockcroft et. al., 198la).
Parakeets (Edwards and Luntz, 1974; Lee et al., 1983), chickens (Kom et.al.y 1968),
turkeys (Boyer et al., 1974), and doves (Cunningham et al., 1976) have all been shown to
precipitate "bird fender's lung" or HP associated with bird serum proteins, although
pigeon-breeder's disease is the best studied (Reed et al., 1965; Stiehm et al., 1966; Stiehm
et al., 1967). Pigeon breeder's disease has been reviewed by Christensen et al. (1975) and
Schmidt et al. (1988). As with other biological antigen-producers, any serum proteins from
any bird could probably cause HP in an appropriate host with appropriate exposure. Multiple
antigens appear to be responsible for pigeon breeder's disease. Some antigens appear
primarily related to exposure, whereas others appear only in the sera of symptomatic
individuals (Berrens and Guikers, 1972; Berrens and Maesen, 1972a; Berrens and Maesen,
1972b). At least five major antigens have been identified and described (Edwards et al.,
1970; Edwards et al., 1969; Fredricks and Tebo, 1975; Tebo et al., 1977).
Mammals produce proteins that can cause HP if aerosolized appropriately in the
presence of susceptible hosts. Most cases of HP associated with mammalian proteins occur in
occupational environments. Laboratory animal handlers, especially those handling rats, are
occasionally affected (Salvaggio, 1987). Hypersensitivity pneumonitis associated with
household mammalian pets has not been reported.
Thermophilic actinomycetes, although common on natural substrates in the outdoor
environment (soil, compost), are not common in air and are rarely recovered from indoor air
unless a contaminated reservoir/disseminator is present. Based on analyses of situations
known to cause HP, it appears that massive exposure to appropriately sized antigens is
necessary for the development of HP. For example, dairy fanners that use moldy hay in
closed barns late in the season are more likely to contract the disease than hog or cattle
ranchers who do not handle contaminated organic material in enclosed spaces (Fink, 1983).
Hypersensitivity pneumonitis, then, is probably a disease of the indoor environment.
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Preliminary evidence indicates that coexposure to an antigen and endotoxin may be more
sensitizing than exposure to an antigen alone. Certainly, gram-negative bacteria are always
present where dead plant materials are handled. Although high levels of antigens are
important for sensitization, it is probable that very low antigen levels will subsequently
induce symptoms and present a risk of continuing lung damage.
Risk factors for development of HP are unknown, but may involve a defect in the
cellular immune system. Pigeon breeders with active disease were shown in one study to
have depressed T suppressor-cell activity when compared with comparably exposed,
asymptomatic breeders (Keller et al.; 1984). The attack rate can be low-in spite of large
populations receiving high levels of exposure to suitable antigens, further^suggesting some
specific host risk factor. The role of these factors may be different when exposure to
antigens is coupled with exposure to endotoxins. Elevated attack rates in some situations may
be explained by this phenomenon.
Because the disease is not expected to occur in so-called clean environments (offices,
homes), and in early stages misdiagnosis is probably common, the actual incidence of HP is
unknown. It is estimated that 7% of British farmers have farmer's lung (Boyd, 1971; Grant
et aL, 1972). Epidemiologic studies in other environments have not been undertaken.
Among clearly exposed populations, attack rates can be as high as 15 to 21 % in offices and
among pigeon breeders, respectively (Banaszak et al., 1970; Caldwell et al., 1973; Emanuel
et al., 1964). .
4.2.2.2 Detection of Sensitization
Serum IgG antibodies~fcs-' detected by the precipitin test, are produfed. in nearly all
people with symptoms and in possibly 50% of exposed people without symptoms (Fink et al.,
1972). The antigen-specific IgG ELJSA is a more sensitive and quantitative assay that may
be used to determine differences in levels of antibody in exposed asymptomatic and
symptomatic people. However, this use has not been reported. Of course, these studies are
only possible where a specific causative antigen has been identified. Tests of cell-mediated
immunity (e.g., pulmonary lymphocyte blastogenesis) define more clearly the differences
between symptomatic and asymtomatic exposed populations, suggesting a primary role for
cellular immune systems in the pathogenesis of the disease (Moore et al., 1980). Challenges"
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(inhalation of antigen aerosols) with known or suspected antigens can be used to reproduce
symptoms and to relate disease to specific antigen sources.
4.3 BIOLOGICAL TOXINS
Toxins can enter the mammalian system by ingestion; absorption through the skin;
inhalation; and subcutaneous, intraperitoneal, or intravenous injection. Toxic effects can be
acute and/or chronic. The toxin may exert its effect on a cellular level at the primary site of
entry (skin, lung, esophagus, stomach), on organs where nontoxic precursors are metabolized
(kidney, liver, bladder), or systemically (neurotoxicity). Most of the biological toxins are
cytotoxic at relatively low doses.- Many are also teratogenic, mutagenic, and/or carcinogenic.
The bacterial toxins have been considered primarily important as part of infectious disease
syndromes (e.g., tetanus) or as ingested poisons (e.g., botulism). Mycotoxins (fungal toxins)
have been primarily studied as ingested poisons (e.g., ergotism, aflatoxin carcinogenesis).
However, it is becoming clear that, when inhaled, mycotoxins may be responsible, either
directly or in conjunction with other agents, for some of the diseases associated with indoor
air quality. Three major kinds of biological toxins will be considered here: the bacterial
endotoxins, the mycotoxins, and fungal volatile organic compounds.
. / " . '
4.3.1 Bacterial Toxins
Bacterial toxins are of two types: exotoxins and endotoxins. Exotoxins are bacterial
metabolites that are excreted into the environment. The toxin produced by.Clostridiwn
bondinum, responsible for the serious form of food poisoning known as boTfuEsm, is a
bacterial exotoxin. Exotoxins have not been studied -with respect to aerosol exposures or
their presence in the environment. "' :
Endotoxins have been the focus of intensive study for many years, especially with
respect-to their role in infectious disease (Westphal et al., 1977; Westphal et al., 1983).
Endotoxins are a component of the outer membrane of gram-negative bacteria and are known,
in pure form, as lipopolysaccharides (LPS). Lipopolysaccharides are composed of three
regions: .'.', * -' - "' ;
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the O-specific polysaccharide chain that is specific for each type of bacterium;
the R-specific core polysaccharide that is relatively similar among bacteria; and
lipid A, which has both constant and variable regions.
The lipid A part of the molecule is responsible for the toxic effects, whereas the
polysaccharide parts constitute the antigens. Lipopolysaccharides are stable and resist routine
autoclaving. In the environment, endotoxins may be part of whole cells, large membrane
fragments, or in macromolecular aggregates of about 1 million daltons (American Conference
of Governmental and Industrial Hygienists, ;1989b).
Endotoxins are primarily known as pyrogens (fever-inducers) and are highly toxic. In
addition to fever, they cause acute pulmonary changes and local inflammatory responses
(Ellakkani et al., 1984; Lantz et ah, 1985). Endotoxins are also nonspecific immune system
stimulants and may be anticarcinogenic (American Conference of Governmental and Industrial
Hygienists, 1989b; Enterline et al., 1985). Environmental exposure to endotoxins occurs in
occupational environments where organic materials contaminated with gram-negative bacteria
are handled. Byssinosis (pulmonary disease related to exposure to cotton dust) lias been
extensively studied withirespect to the role of endotoxin (Castellan et al., 1987; Rylander
et al., 1985; EUakkani et al., 1984; Cinkotai et al., 1977; Kennedy et al., 1987). Poultry
and swine confinement buildings (Clark et al., 1983b), composting facilities (Clark et al.,
1983a), grain elevators (DeLucca et al., 1984), and environments where gram-negative
bacteria are used in manufacturing processes (Palchak et al., 1988) all present a significant
risk for exposure to endotoxin aerosols. In addition, humidification equipment, including
home humidifiers, are frequently .contaminated with gram_-negative bacteria and, hence, with...
endotoxins. Some evidence indicates that symptoms associated with the operation of
contaminated humidifiers result from endotoxin exposure and direct activation of the
inflammatory process via the alternate complement pathway rather than through the agency of
an antigen (Rylander et al., 1978; Rylander and Haglind, 1984). Alternatively, the endotoxin
might be acting as an adjuvant. These hypotheses might explain the frequently high attack
rate that has been reported in some epidemics of humidifier fever as compared to the much
lower rates that prevail for farmer's lung disease and the frequent lack of association of
precipitating antibodies to suspected source material in exposed people. Endotoxin may be an
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auxiliary factor in immunologic sensitization for asthma as well, although this hypothesis
remains speculative (Michel et al., 1989).
Doses of endotoxin that have caused symptoms in the environment are unknown.
Measured levels in the environment range from 0 to > 100 jig/m3, with 0.1 to 5 fig/mL
measured in contaminated humidifiers associated with disease (Rylander and Haglind, 1984).
Quantitative endotoxin data must be interpreted with daution because values are strongly
dependent on the method of assay. The assays for endotoxins that are currently available are
comparative rather than absolute and do not allow comparisons between laboratories (Milton
etal., 1990).
4.3.2 Mycotoxins
Most fungi produce metabolites that nave a range of toxic effects ranging from mild
acute toxicity to potent carcinogenicity (Shank, 1981; Rodricks et al., 1977). Taxa such,as
Penicillium, Fusarium, and Aspergillus, members of which produce toxins that have dramatic
acute effects (e.g., aflatoxins, antibiotics, trichothecenes), have been extensively surveyed for
toxin production, whereas other taxa have not been well studied (Surge, 1987). Some fungal
' - f-
toxins are toxic without metabolic activity (e.g., T-2 toxin),- whereas others require metabolic
conversion for toxicity (e.g., afiatoxin Bj). Fungal toxins that require conversion often
exhibit their toxic potential in target organs able to effect the metabolic conversion (e.g.,
liver). Although little studied, lung tissue can apparently convert afiatoxin BI} and probably
other mycotoxins, to their toxic form. Most of the mycotoxins are cytptoxic as measured in
cell culture in the range of 0.1 to 10 jig.of toxin/mL of culture fluid.
Mycotoxins can enter the body through the skin (Riley et al., 1985), gastrointestinal
tract (Shank, 1981), or respiratory tract (Wicklow and Shotwell, 1983). Mycotoxins become
airborne or aerosolized on substrate material, adsorbed onto dust particles or spore/mycelial
surfaces, or as an intrinsic part of spores or mycelial fragments (Wicklow and Shotwell,
1983). Therefore, the toxins are carried on a wide range of particle sizes with different
degrees of penetrability into the respiratory tract. The smallest particles may reach alveoli in
significant mass, larger particles may deposit in the conducting airways, and the largest are
probably held in the nose or trachea and subsequently swallowed. Respirable dust particles
that remain on lung surfaces for some-time present a greater dose directly to lung tissue with
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a resultant increase in the risk of local tissue damage and possible neoplasm development
(Baxter et al., 1981). In addition, inflammation has been shown to be a cofactor in
carcinogenesis so that toxins borne on potentially inflammatory dusts (e.g., grain dusts and
other particles containing endotoxin) may be carcinogenic in very low doses (doPico et al.,
1977).
Acute toxic effects from airborne mycotoxins are rarely reported, but do occur. Severe
acute toxic effects have been reported from exposure to a massively contaminated return air
duct containing the trichothecene-producing fungus Stachybotrys aim (Croft et al., 1986).
"Yellow rain," a biological warfare agent, used in southeast Asia was thought to be
concentrated trichothecenes (Miroeha et al., 1983). Tremorgenic mycotoxins produced by
- ' -*£ .'
Aspergillus jumigams may have caused occupational symptoms in a sawmill environment
(Land et al., 1987). Acute symptoms experienced by grain handlers may be, in part, due to
the mycotoxins as well as lectins and other toxic agents in grain dust (doPico et al., 1977,
1982, 1983; Dashek et al., 1983; Palmgren et al., 1983; Enarson et al., 1985). It has been
suggested that some of the symptoms mimicking sick building syndrome (headache, dizziness,
nausea, fatigue) may be due to exposure to airborne trichothecenes. Mycotoxins njay also be
responsible for the pathogenesis of invasive fungus diseases such as aspergillosis, the toxin
paving the way for fungus invasion (Hchner and Mullbacher, 1984).
Chronic exposure to airborne mycotoxins also represents a significant health risk.
Cancer, probably associated with low-level mycotoxin exposure, has been reported in peanut
handlers (Burg et al., 1981, 1982), mycotoxin researchers (Deger, 1976), and in several
farm-related cases (Dvorackova and Pichova, 1986). Reports of "leukemia houses" are
ti;.;,.
thought to be the result of afialoxin exposure (Wray and O'Steen, 1975): Three cases of
pulmonary interstitial fibrosis have been reported where aflatoxin Bj was measured in lung
tissue on autopsy (Dvorackova and Pichova, 1986).
Analysis of air samples for mycotoxins is usually restricted to the aflatoxins, or
occasionally, the trichothecenes. Aflatoxins have been recovered from airborne grain dust in
levels exceeding 1,800 parts per billion (Burg et al., 1981; Burg and Shotwell, 1984; Dashek
et al., 1983; Palmgren et al., 1983; Sorenson et al., 1981). For aflatoxins, particles less
than 5 jim appear to carry the preponderance of toxin (Sorenson et al., 1981). Sampling
methods used for airborne toxins were not designed for particles less than 0.1 pun, and toxins
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on these small particles have not been studied. More importantly, considering the high
potential for synergism between toxins, most studies have examined only single toxins. It is
clear that many fungi produce multiple toxins and that many different toxigenic fungi may
inhabit specific environments. The potential for exposure to unknown toxins must be
assessed. For example, farmers harvesting corn are not only exposed to aflatoxins and
trichothecenes, but to more than 1010 fungus spores per cubic meter of air, including known
toxin producers such as Altemaria, Cladosporium, Penidllium, and Aspergillus. Acute or
chronic exposure to any mixed microbial aerosol presents some risk of toxin exposure.
4.3.3 Fungal Volatile Organic Compounds
In addition to higher molecular weight ioxins, all organisms exude volatile organic
compounds (VOCs) during growth. Although these VOCs are gases, due to their source,
they are closely involved with bioaerosol-related problems. Some of these biological volatiles
impart characteristic odors to the environment. Examples of such odors include body odor
produced by human occupants of interior spaces; dirty sock odor from bacterial growth on
damp, sweaty clothing and carpeting; and the moldy smell, characteristid of some basements^
Some of the VOCs that have been reported from fungi grown on "natural" substrate
(wheat meal) include 3-methylbutanol, 3-octanone, 3-octanol, lrocten-3-ol, 1-octanol, and
czs-2-octen-l-ol (KaminsM et ah, 1972; Kaminski et al., 1974). Penicillium species grown
on potato dextrose agar produces thujopsene, 3-octanone, nerolidol, l-octen-3-ol, phenylethyl
alcohol, 3 octenol, l,5-octadien-3-one, l,5-octadien-3-ol, 2-methbxy-3-isopropyl-pyrazine,
2-methylisobomeol, 2-methyl-l-propanol, 2-methyl-2-pentenal,, 3-methyl-l-butanol,
naphtalene, damascenone,. and octanoic acid (Halim et al., 1975; Karahadian et al., 1985).
Penicillium chrysogenum grown on malt extract agar produces many of these same
compounds and, in addition, many compounds that have been reported from building
materials off-gassing in the indoor environment (unpublished data). Some of these reported
compounds (e.g., acetaldehyde, heptane, 2-heptanone, 2-hexanone, 2-pentanone, etc.) may
produce significant adverse health effects at sufficiently high concentrations. Others produce
strong odors (e.g., decanoic acid, hexanoic acid, methanethiol). In addition, fungi are able;
to metabolize toxic solids into a gaseous state. For example, some fungi can convert arsenic
compounds into trimethyl arsine (Foster, 1949) and have resulted in arsenic poisoning. Many
/ 4-27 :
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fungal VOCs produce mucous membrane irritation and may be involved in or produce
symptoms (headache, nausea, dizziness) that mimic the sick building syndrome.
Unfortunately, very little research has been done to characterize microbial VOCs, assess their
exposures, or evaluate their potential health effects.
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5. BIO AEROSOL INVESTIGATIONS
Bioaerosol investigations, whether for research purposes or to solve specific problems,
rely on a knowledge of the principles of aerobiology, air sampling technology and analysis,
as well as epidemiology, and a thorough familiarity with the nature of bioaerosols.
Unfortunately, this combination of knowledge cannot be obtained in any single discipline.
Those who do bioaerosol research effectively operate in a team environment. Research
investigators approach;questions related to bioaerosols differently than field investigators.
However the same factors must be considered:
investigative strategy,
sample collection techniques, -
sample analysis methods, and - '
data analysis and interpretation approaches. .
5.1 Investigative Strategies
Nonbiological pollutants in the work place are easy to assess, relative to biological
pollutants. Methods for aerosols, and standards or guidelines are available that allow
interpretation of collected data (American Conference of Governmental and Industrial
Hygienists, 1984).' For bioaerosols, this simplified situation is rarely the case-.: Because
available sampling methods have intrinsic differences and require skilled 'and time-intensive
analysis, little background data exists on indoor bioaerosols, and no guidelines have been
published. Thus, relatively intensive investigations are usually required, and difficult
decisions must be made with respect to sampling strategy. .
The total air environment in any situation cannot be sampled. Rather, a statistically
adequate sample must be collected and overall patterns of exposure must be inferred.
A necessary or suitable size sample has not been determined for bioaerosols. Such a
determination would require that multiple duplicate samples be taken with several different
kinds of equipment. The samples would also have to be taken at many sites in the
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environment and over a time period that included all the potential variability of the predicted
aerosols.
5.1.1 Studying Indoor Microbial Ecology with Respect to Air Pollution
At the most basic level, indoor bioaerosols may be considered as an ecosystem. In this
case, human exposure is not the concern, but rather factors controlling population dynamics
of sources, the dynamics of dissemination, and the dynamics and biology of airborne
populations are the concern. These studies provide the basis for all bioaerosol investigations.
. A few of the factors that must be considered are
. the role of environmental factors (substrate, climate) on microbial survival, growth,
reproduction, and metabolism;
factors controlling dissemination from reservoirs; and
. the taxonomy of environmental microbes (Dimmick and Akers, 1969).
5.1.2 Documenting Exposure/Dose/Symptom Relationships
To set standards or develop guidelines for interpretation of bioaerosol data, exposure-
response information is necessary. These data may be generated in the laboratory from
animal or human exposure studies, or epidemiologicaUy, by collecting baseline data and
evaluating symptoms in a wide variety of environments. Documenting exposure requires air
sampling and assessment of particle size as well as other exposure parameters.
Questionnaires and/or^Mal exams may be used to evaluate symptojg^Su et al., 1990;
Burge and Garrison, 1989). -^F^'
5.1.3 Documenting Unusual Exposure Situations
Documenting unusual exposure situations is the simplest approach to evaluating
bioaerosol exposures and is usually appropriate for problem-solving investigations done in
response to epidemics of disease or building-related symptoms. Field investigations designed
to approach specific building-related problems may be divided into two steps: a careful
analysis of the biological status of the environment and an evaluation of the environmental _
data with respect to health risks.
5-2
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Ideally, air sampling should be used to assess airborne exposure levels. Studies that
include air sampling need to be designed with appropriate controls, and need to include
enough samples so that reasonably valid interpretations may be made. Minimally, it is
essential to have duplicate samples in the environment of symptomatic people, asymptomatic
people in the same environment, and samples of the outdoor air near air intakes. Sampling
before and after disturbing potential reservoirs/amplifiers is also extremely useful.
A second approach involves searching for potential reservoirs/amplifiers, assessing
^ contamination (visually or .through assay techniques), and either providing logical hypotheses
for dissemination/exposure or conducting air sampling in association with postulated
dissemination conditions and comparing air sample results to those obtained with samples
from the potential source. Concluding that an unusual exposure situation exists is relatively
straightforward if high levels of an organism, antigen, or toxin are found in indoor air and
not outdoors or in other control situations. Unfortunately, proving the negative case is
.rarely, if ever, possible even when sampling protocols are multifaceted and extensive
(American Conference of Governmentaland Industrial Hygienists, 1989b).
5.2 AIR SAMPLE COLLECTION TECHNOLOGY
Air sampling for biological aerosols is usually done to estimate the potential for human
exposure. Occasionally, bioaerosol sampling is done for legal reasons. In both cases, but
especially in the latter, it is essential that_ the sampling procedures can withstand legal,
challenge. This means that either the best, available sampling strategy,.instrumentation and /,.
methods for analysis, must be used or one must be able to document the validity of using less
efficient or less reliable methods. /
: - Sampling can be either personal or ambient. Personal sampling (i.e., sampling near the
breathing zone of affected individuals) would be ideal. However, personal samplers that
reliably estimate most microbial aerosols have yet to be developed .(American Conference of
Governmental and Industrial Hygienists, 1989a). Ambient sampling is most often used for
both microbial and antig'en aerosols. Ambient sampling, if properly done, reflects average
exposures for all occupants, but can underestimate individual doses. To be effective, ambient.
sampling must be done at all the levels and sites where people work or live and over a time
: ' 5-3- .
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period that covers potential cyclic changes in the aerosols and activity patterns of the people
(American Conference of Governmental and Industrial Hygienists, 1989a).
Air sampling may be performed either over short periods (grab samples) or over longer
periods (average or integrated samples). Grab samples usually represent 5 minutes or less of
sampling time, whereas integrated samples are collected over a longer exposure period. All
of the methods commonly used for viable microorganisms collect grab samples. It is difficult
to maintain viability and/or prevent growth of viable microorganisms over longer sampling
periods.
Size-selective sampling has always been considered important for bioaerosol-related
studies because lung deposition has been considered necessary for efficient transmission of
infectious disease. However, upper airway deposition of allergens clearly causes disease
(Wilson et al., 1973). Except in research situations, most microbial samples can be collected
as a unit on a single-stage sampling device. At most, two stages are useful for determining
the so-called "respirable" fraction of the aerosol.
Accuracy and precision are factors that apply to all aerosol sampling methods..
Accuracy is the amount by which the measured value deviates from the true value.^.P.recision
is the standard deviation of repeated measurements of the same variable. Accuracy is
difficult to assess for most bioaerosols. For chemicals, accuracy of a particular method is
measured by using another method and comparing results. Chamber studies that provide a
continuously stable aerosol are necessary for most bioaerosols. Precision can be assessed by
increasing the number of measurements at a particular site using the same methodology.
5.2.1 Principles of Aerosol Sampling -&&&
The term aerosol technically refers to the complex of particles suspended in a gaseous
medium, although the popularly used definition is synonymous with "particle". Bioaerosols,
then, are airborne particles with a biological origin that follow the physical rules for any
aerosol. Principles of aerosol sampling have been reviewed elsewhere (Edmonds, 1979).
Aerosols can be collected using gravitation, centrifugal force, inertial impaction, filtration,
and electrostatic impaction (American Conference of Governmental and Industrial Hygienists,
1989a; Burge and Solomon 1987). The most commonly used modalities for collection of
bioaerosols are inertial impaction and filtration. Regardless of the sampling mode, when
5-4
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selecting sampling devices for biological aerosols, the efficiency with which particles are
removed or collected from the aiiy the efficiency with which viability is protected, and the
overall efficiency must be considered (American Conference of Governmental and Industrial
Hygienists, 1989a). \
, Inertia! impactors collect samples by drawing air through an orifice using suction (e.g.,
the sieve-plate, slit, and centrifugal impactors) or by rotating collecting surfaces rapidly
through the air (e.g., rotorods). Suction devices require that the ambient airflow be directed
into the sampler orifice, and that the rate of suction be equal to the ambient air speed. This
is called isokinetic sampling. . Wherr suction sampling is not isokinetic, patterns of particle
/collection change. For example, if suction speed is greater" than air speed, small particles
will tend to be vacuumed from the air and will be over-represented in the sample (May,
1967; May, 1980). Similar kinds.of effects occur if the sampler orifice is not directed
towards the airflow. In the extreme case where the orifice is at right angles to the ambient
airflow, smaller particles are likely to be preferentially sampled at all suction speeds (Akers
and Won, 1969). In general, in most indoor environments, an isokinetic-sampling condition
can be ignored if the aerosol of interest is less than 10 /im and sampling rates (airflow
through the sampling orifice) are low. Errors may be introduced when larger aerosols are
being sampled. Many common allergens, including all pollen and many fungus spores, are
larger than 10 ^m. . ,
Once the particles are in the sampler, impaction efficiency depends on the inertia of the
particle as it hits the impaction surface. Larger particles have more inertia at a given particle
speed and are more likely, to impact. Thus, although suction samplers readily draw small
spores into the sampling-.orifice, larger particles are more likely to impact on collection
surfaces and be counted. Only a few samplers have been well characterized with respect both
to aerosol-collection efficiency and particle-retrieval efficiency. Those that have not should
be used with care.
Filtration devices also rely on suction for collection of the aerosol. Once in the device,
theoretically, filtration samplers are 100% efficient at trapping particles larger than the rated
.filter pore size. Therefore, if suction conditions are chosen appropriately, very high sampling
efficiency may be obtained using filtration.
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The width of the collection surface controls the efficiency of small particle collection for
the rotating impactors. Unless the collection surfaces are well under 1 mm in diameter, small
particles are poorly collected by these devices (Edmonds, 1972).
5.2.2 Sampling for Viable Microorganisms
1" i1
Some organisms must remain viable during sampling and analysis procedures. Many
infectious agents, including all bacteria and many fungi, are only identifiable in the living
state. Factors that affect viability in the air of these kinds of aerosols are covered elsewhere
' in this document. However, the process of sample collection, handling,_and analysis also
affects viability or culturability.
Sample collection can damage living cells in many ways, including mechanical effects
caused by impaction and desiccation, as demonstrated by a decrease in bacterial recovery
when slit-to-agar distance is decreased in a slit sampler, or when the slit width is decreased.
The speed at which the particle hits the agar surface is increased by both of these changes and
- the losses are presumed due to mechanical breakage of the cells (Goldberg and Shechmeister,
1951). The effects of changes in relative humidity on microorganisms are complex and
>x jpoorly studied. It has been shown that bacterial cells collected on a dry filter rapidly
"'' '"desiccate with'resnltingiiQss^in^viability (Wolochow, 1958). In addition, rehydration of
aerosols of bacteria and viruses may cause cell damage (Hatch and Dimmick, 1966; Akers
et al., 1966). Neither of these effects have been well studied for fungi.
It should be apparent that the media into which ceUs are collected should not adversely
impact viability. Wetting agents, growth inhibitors, antibacterial, arid/or antifungal agents all
* . r-jfty?**'**"*
have adverse effects on romeiorganisms (Surge et al., 1977b). In addtop, culture media
must be chosen that either will support the growth of the organism directly, or will protect its
viability until transfer to appropriate media can be undertaken. In general, rich media (e.g.,
nutrient or casein soy peptone agar for bacteria, malt extract agar for fungi) are used in air
samplers. However, use of minimal media may allow stressed organisms to recover, and
recoveries will vary depending on the culture medium used (Akers and Won, 1969). The
cultural sampling data is in the form "colony-forming units" rather than spores or cells unless
dilution methods (e.g:,-liquid impingers) are used. There is no way of knowing how many
original cells contributed to a single colony on a culture plate impactor.
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; Culture plate impactors (preseeded with living cells for viruses), liquid impingers,
membrane filter cassettes, and high-volume electrostatic devices have been used to collect
viable microorganisms. Viruses, infectious bacteria, fungi, and protozoa are usually sampled
by methods that allow their culture both in vitro and in vivo. Therefore, viability is of
concern. Although viruses are submicronic, they usually travel through the air on larger
particles and the same sampling devices may be used as for larger bacteria and fungi.
Bacteria also tend to travel on rafts (masses of organic material). Indoor human-source
bacteria generally travel on particles larger than 20 pm. The thermophilic actinomycetes and
the fungi, on the other hand, appear to travel as single spores and may be as small as 1 to
2 pm.
When choosing a sampler for collection of a particular viable aerosol, the size of the
particles, the relative fragility of the organisms, and the expected concentrations should be
considered. For very small particles (small fungus spores, thermophilic actinomycetes),
suction devices and isokinetic sampling should be used and impactiori efficiency within the
sampler should be well characterized. Larger particles also require isokinetio conditions if
suction sampling is used. However, impaction efficiency becomes somewhat less of a
concern.
A hypothetical "order of decreasing fragility" for common indoor microbial aerosols is
likely protozoa > vegetative bacterial cells > viruses > fungal spores > bacterial endospores
(including thermophilic actinomycete spores). Therefore, relatively gentle methods should be
used for protozoa and vegetative bacteria, whereas more aggressive techniques may be used
for fungal spores and bacterial endospores. In practice, the same sampling devices are
generally used for all, and careful consideration of the relative potential for loss of viability
must be taken when the more fragile organisms are sampled.
Intercellular interactions are well known for fungi and have also been reported for
bacteria. It is clear that the more fungal spores that impact on an agar surface, the fewer will
germinate and produce a recognizable colony. Therefore, it is essential to select sampling
rates and volumes that will not overload the sampling surface when using a culture plate
impactor. In addition, it should be noted that some fungi and actinomycetes can produce
diffusible antibiotics that will kill many bacteria and some fungi. When sampling dense
aerosols of any type, it .is probably advisable to use a method where dilution culture can
5-7
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easily be used to minimize damage from these compounds (e.g., liquid impingers or filter
cassettes).
5.2.3 Sampling for Microscopically Identifiable Organisms
Both bacteria and fungi can be sampled by methods that allow for direct microscopic
examination. These methods produce total counts of individual cells in contrast to viable
colony-forming units. Commonly used methods include suction slit impactors, membrane
filter cassette samplers, and rotating arm impactors. The suction sampler readily
overestimates small aerosolized particles in still air, and underestimates them in air moving
. . v'i^j:
faster than the sampler flow rate (May, 1967). However, because viability; is not a concern,
samples can be collected over long periods of time for analysis by direct microscopy. Thus,
one slit impactor collects a time-discriminated sample over a 7-day period (the Burkard
version of the Hirst spore trap). Filter cassettes can be used at low flow rates over an entire
work shift, allowing integrated 8-hour exposure assessments to be made.
The rotating arm impactors are the most commonly used samplers in the United States
for outdoor bioaerosols (with emphasis on pollen). They are highly efficient at collecting
particles in the size range of most pollen and are easy to use. However, if the collecting
surface is > 1.5-/tm wide, they are less than 5% efficient for fungal spores smaller than 5
(Edmonds, 1972). In dense viral aerosols where infectivity is not of concern, it is also
possible to collect the samples on a membrane filter for subsequent examination by electron
microscopy. Some interest has centered on the use of filter cassettes with subsequent direct
microscopic examination for total bacteria and/or fungi or with elution and culture. Bacteria
must be stained for microscopic analysis. However, these methods appear ,to underestimate
both viable and nonviable aerosol concentrations. They are useful for enSdtoxins, and
possibly for other toxins and allergens, provided sample volumes are of sufficient size.
5.2.4 Sampling for Amorphous Particles
Many of the allergens, antigens, and toxins that may accumulate in the indoor
environment are carried on particles that do not grow in culture and are not readily
identifiable microscopically. Collection of these particles from air, although requiring
adherence to the same aerosol collection rules that apply to other particles, also must enable
5-8
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the particles themselves, the soluble adhering material, or other associated material to be
eluted for assay or assayed directly from the sampling medium. For water soluble materials
that are not hydrophobic, liquid impingement is a possible choice. Most investigators,
however, use some kind of filter medium in a suction sampler. For endotoxins, smooth
surface polycarbonate filters minimize the risk of permanent adherence of the toxins to filter
surfaces (Milton et al., 1990). Adherence of other materials to filters has not been carefully
tested.
Mite, cat, and small mammal antigens have all been collected from indoor air using
high-volume filtration devices .(Swanson et al., 1985; Plarts-Mills et al., 1989). The suction
rate used in these devices is high and small particles are preferentially collected. It is also
probable that the high suction rate changes the environment by, in effect, cleaning the air.
These two factors make high-volume filtration sampling in indoor air a qualitative rather than
a quantitative procedure.
5.2.5 Sampling for Volatile Aerosols
. ^
Both fungi and bacteria produce volatile compounds that are odoriferous and may cause
irritation and other health risks. Sampling for these compounds requires prior knowledge of
their nature. Water soluble volatiles are collectible with a liquid impinger. Various
adsorbents (e.g., charcoal, tenax) may be used for some volatiles. Cold traps (i.e., liquid
nitrogen) are used to collect a total volatile component. These methods, however, have not
yet been applied to microbial volatiles in.other than research settings.
5.2.6 Source Sampling
Source sampling involves collecting materials from suspected reservoirs for analysis
using any of the methods discussed below (e.g., culture, microscopic observation,
immunoassay, biological assay, or chemical assays). Reservoirs that are commonly assessed
include house dust, fluid reservoirs, soft materials, and surfaces.
House dust mite allergens, cockroach allergens, fungi, .and bacteria are commonly
assayed from samples of house dust collected with vacuum devices (Gravesen, 1978;
Platts-Mills et al., 1986b). In general, principles of aerosol collection have been ignored in
these studies. Results are presented as antigen, mites, or colony-forming units of fungi or
. -, -. '' , V". ' 5-9 . , ' '.'. "."
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bacteria per gram of dust. Unfortunately, the size of particles collected as well as the
location within the reservoir (i.e., carpeting) is controlled by the amount of suction at the
vacuum orifice, and the size of particles retained depends on the pore size of the collection
bag. As a result, although individual studies using a single vacuum device allow comparisons
from site to site, studies using different collection devices are not comparable. Also, results
using poorly characterized collectors do not accurately represent the potential for exposure.
Sampling from fluid'reservoirs entails collecting the sample (water/slime) in a. bottle or
syringe for subsequent analysis. Where water samples are concerned, a reasonably valid
sample can be obtained if the reservoir is weU mixed prior to sample collection. Slime that
collects on wet surfaces can be scraped into a collection container. These samples are likely
to represent local conditions; however, multiple sites should be sampled to assess reliability.
Soft materials such as carpeting and other fabrics, fiberglass and wall paper, and other
paper materials may be collected for microscopic examination or elution for other assay
methods. Obtaining a representative sample is a considerable challenge. Often one collects
parts of these materials supporting visible fungi growth only for determination of the kind of
fungi and not the quantity present.
Where removable, hard materials can be taken to the laboratory for analysis. Usually,
however, scrapings must be collected (from areas of obvious growth) or in situ surface
samples must be taken. Transparent tape pressed against obvious contaminated surfaces
allows straightforward microscopic examination. The tape can be dissolved with acetone
without damaging collected spores. Surface cultures can be obtained either using sterile
swabs or contact plates. Neither of these methods is quantitative, although they are often
^5^ "
used by inexperienced investigators to evaluate levels of contamination. ^Obtaining a
$?#"**' . '
representative sample, especially of organisms growing on the surface (father than having
settled), is nearly impossible. Table 5-1 briefly summarizes sampling modalities commonly
used to evaluate biological contamination of the indoor environment.
5.3 AIR-AND SOURCE-SAMPLE ANALYSIS METHODS
Although sample collection principles are common to both biological and nonbiological
aerosols, analysis methods are quite different and require procedures specific to each different
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TABLE 5-1. SUMMARY OF SAMPLING MODALITIES USEFUL
FOR INDOOR BIOAEROSOLS
Method
Cultural
Impaction
Impingement
Direct Visualization
Impaction
Filtration
Impingement
Chemical Assay
Filtration
Impingement
Viruses
X
X
t
X
' X :
,
Bacteria
X
X
X
X
-
, Fungi
x
; ' -
X
X
Pollen Antigens Toxins
^ r " ,
X - .;' '
x ' .
X\r
""*
XX
bioaerosol. Bioaerosol analysis methods fall into five general categories: direct microscopy,
culture, immunoassay, bioassays, and chemical analysis. -
5.3.1 Direct Microscopy
Fungi spores can be counted microscopically from any sample that either is collected .
onto an optically suitable surface (transparent tape, microscope slide, plastic rotorod), onto a
surface that can be made transparent (some filter media), or into a medium.that can
subsequently be filtered (e.g., a liquid impinger). Many fungal spores are identifiable using
microscopic examination without staining. Bacteria, including Legionella, are countable
microscopically only if stained., The type of stain used depends-on the chemical nature of the
organism (e.g., .acridine orange fluorescent staining for bacteria, basic fuschsin or
phenosaffranin stains for pollen) (Palmgren et alM 1986; Muilenberg, 1989), or the antigenic
composition (e.g., fluorescent antibody stains used for Legionella) (Winn, 1985). Most
particle collection methods can be used with subsequent staining, providing the impaction
medium (often silicone grease), when present, does not interfere with the staining. Although
pollen can readily be stained after impaction in grease on a rotorod or Burkard trap slide,
acridine orange and fluorescent antibody stains do not penetrate well through a layer of
grease. For these, filter collection media or liquid collection are more appropriate.
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Scanning electron microscopy (SEM) must be used to visualize viruses. Although
useful for determining surface characteristics, SEM does not usually significantly add to the
information necessary for bacterial or fungal spore identification.
Although attempts have been made to count spores and pollen using digitized
microscopic images fed into computer programs, so far these methods are impractical because
of the enormous morphological variability in most bioaerosols. Likewise, particle counters,
including those using advanced laser techniques, do not allow differentiation between the
bioaerosol fraction and the total particulate fraction in air samples.
5.3.2 Culture %&
Many viable organisms (fungi and bacteria) that are collected by impactibn on a culture
plate surface can reproduce and produce visible colonies. Each colony may represent one or
more original cells, so that one colony is called a colony-forming unit. This is in contrast to
liquid impingers, where cells are usually separated into single units before culture.. Culture
plate impaction methods will always underestimate actual airborne cell counts both because of
this possible multiple cell bias and because not all airborne cells are ever viable and no single
medium will support the growth of all organisms! the conditions of incubation must be
appropriate and the culture medium must not be overloaded.
For air sampling, relatively rich culture media are-generally used. These media usually
contain necessary carbohydrates in a simple form (e.g., glucose) and amino acids in a natural
form (e.g., peptone, blood, soy digests, etc.). On these media most healthy organisms will
grow, given time. The organisms actually recovered, however, are those, that grow the most
rapidly. Organisms thafhaverbeen damaged before sampling (i.e., stressed organisms) often
begin growth very slowly and are overrun on rich media. For these, very restrictive and
dilute culture media, which slow the growth of all organisms, should be used. If an
organism that is able to utilize an unusual carbon source is suspected, a culture medium
providing only that source may allow that organism to compete more successfully. For
example, Stachybotrys atra, a relatively common toxigenic fungus, grows very slowly in
culture and may not be efficiently recovered when other organisms are also present in a
sample. However, S. 'atra can utilize pure cellulose and culture media containing cellulose as
the sole carbohydrate source provide a competitive advantage to Stachybotrys.
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Another means for reducing competition between organisms is to add biocides that
prevent the growth of particular organisms. Streptomycin and rose bengal are often added to
media designed to recover fungi from environments heavily contaminated with bacteria. It
should be noted that rose bengal becomes a general biocide and will kill fungi, as well as
bacteria, if it becomes light-activated (Burge et al., 19T7b).
Culturing of viruses requires that living cells be present on the culture medium. For
bacterial viruses, bacteria are spread on the plate before the viruses are introduced. For
human viruses, human cell cultures are required. Viruses are usually incubated at 37 °C
(Fields, 1986).
Bacteria can usually grow on nutrient media, although some are fastidious and require
highly specific and complex conditions (e.g., LegumeOa). Most bacteria will grow at 30 °C,
a few are thermophilic, and some can grow at room temperature and below. Incubation
temperatures for bacteria depend on the source. Bacteria that apparently were growing in a
cold water reservoir should be incubated at room temperature. Human source bacteria are
often incubated at 35 °C. The thermophilic bacteria, including the actinomycetes, require a
temperature of 56 to 60 °C. Identification of bacterial taxa .requires Gram-staining,
microscopic examination, and several physiological tests (e.g., production of oxidase and
catalase). For some bacteria (including Legionella), fluorescent antibodies are available and
can be used to microscopically identify species (American Conference of Governmental and
Industrial Hygienists, 1989b).
Fungi will also grow on artificial nutrient media. Fungal culture medium is usually
more acidic than that preferred by most bacteria. Media that will support the growth of
many environmental.bacteria-,are usually.-not:good for fungi and vice versa:';Most fungi grow
best at room temperature. A few can compete well at colder temperatures (psychrophiles).
,-, Most of the fungi that are able to invade human tissue are thermotolerant (i.e., they can grow
at a wide range of temperatures). A few fungi are thermophilic, requiring temperatures in
excess of 45 °G for growth (Cooney and Emerson, 1964). Fungi are identified by their gross
appearance in culture and by the morphology of spore production (Barnett and Hunter, 1987).
Amoebae can be grown in culture if they are provided with a living nutrient source
(usually bacteria). It is thought that amoebae ingest Legionella, which remains alive and
infectious inside the amoebal cell and is protected from the action of biocides. Amoebae are
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identified by their morphology and ability to cause disease when injected into a suitable host.
Fluorescent antibodies are also available for identifying some amoeba species.
5.3.3 Immunoassay
Proteins derived from such sources as cats, cockroaches, and dust mites can be
measured by immunoassay. Most immunoassays are based on extraction of the water-soluble
fraction of a specific antigen source (i.e., each immunoassay is specific for the antigens used
to develop the assay). For example, mite antigen assays are specific only for mite antigens
that are soluble in the systems used, and that will attach to the solid phas|used. Recent
developments allow assays of antigen materials electrically transferred to a solid substrate
without initial water extraction (immunoblotting) (Hoyer et al., 1990). Three types of
immunoassays are currently available:
(1) Radioallergosorbent test (RAST) or ELISA inhibition assays,
(2) direct RAST or ELISA assays, and
(3) immunoblot assays.
\
The RAST and ELISA inhibition assays involve coating a solid phase, usually paper
discs, cellulose beads, or microtiter plates, with a water extract of a known antigen source
and mixing the same antigen (in a series of dilutions) or an unknown sample with human
serum that contains antibodies against the specific antigen. The serum/antigen mixture is
incubated with the solid phase, and the solid phase is examined for human antibodies using an
anti-human antibody labelled1,with a radioisotope (RAST) or an enzyme .gISA) (Agarwal
et al., 1981; Jensen et al., 1989; Swanson et al., 1985). These techniques only require an
allergen extract and serum from one or a group of individuals who are highly allergic to the
relevant allergen. However, these assays are very difficult to standardize and, because the
units are arbitrary, the results for one allergen cannot be compared with those for another
allergen.
Direct RAST and ELISA assays depend on the biochemical purification of specific
antigens from a given source and the development of monoclonal antibodies against each
specific antigen. The monoclonal antibody is then attached to the solid phase, incubated with
5-14 ' .
-------
both known quantities of the relevant antigen and with unknown samples, and then incubated
with a second monoclonal to the same antigen labelled as for the inhibition assays. The
assumption is that the quantity of one protein will act as a guide to the presence of that
source. It is implied that the protein being measured is in a reasonably constant relationship
to other proteins from the same source. In addition, it is necessary that the protein being
assayed be reasonably stable under the conditions of collection and storage. There are two
main advantages of specific allergen assays. First, because the assays can be adapted to give
results in absolute units (i.e., miciograms), the results for one protein can be compared with
proteins from another allergen source. Second, the reagents are sufficiently simple to allow
complete standardization of the assay. Thus, measurements of specific major allergens can be
made in micrograms and st?n
-------
colored precipitate which is visible to the eye and can be optically scanned for quantitation
(Hoyeretal., 1990).
5.3.4 Bioassays and Chemical Analysis
Most commonly used assays for endotoxin depend on the fact that endotoxin causes a
dose-dependent gel reaction in horseshoe crab (Limulus) amoebocytes. Three general types of
Limulus (LAL) assays are available (Jacobs, 1989): the gel-clot method, the turbidimetric-
kinetic assay, and the chromogenic assay. The gel-clot method is simple, more or less
qualitative, and depends on the formation of gel in a tube. The turbidimetric-kinetic assay
evaluates the rate of the gel reaction, as measured by carefully controlled densitometer
readings. The chromogenic assay depends on the production of a pigment released from a
substrate by the clotting enzyme of LAL. Recent developments have included mathematical
approaches to assay analysis that make the LAL assay more sensitive and reliable
(Milton et al., 1990). The assays also measure cell-bound endotbxins less well than free
endotoxins. Unfortunately, in many cases, most exposures to endotoxins may be related to
. intact bacterial cells. For example, although cotton dust is known to contain high levels of
gram-negative bacteria as well as endotoxin, human symptoms correlate more closely with
bacterial levels than with measured endotoxin levels. For this reason, assessment of gram-
negative bacterial levels may be an appropriate surrogate until more accurate methods for
measuring endotoxins have been developed.
Bioassays are necessary to determine pathogenicity of specific strains of infectious
viruses, bacteria, and fungi. Sentinel animals have been.used to document the presence of -
infectious Legionetta. Eavironmental isolates of Hisioplasma do not do well in culture until
they have been passed through a living organism.
Thin layer or high pressure liquid chromatography are used for the detection of many
mycotoxins, including the afiatoxins and the macrocyclic trichothecenes (e.g., satratoxins)
(Shank, 1981). Gas/liquid chromatography .and mass spectroscopy (GC/MS), used for the
Fusarium toxins, is being investigated as a tool for endotoxin analysis and is the method of
choice for volatile organic compounds derived from microbial growth. All of these methods
are readily applicable to air samples, but have not been extensively used.
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Attempts have been made to biochemically "fingerprint" specific bacterial taxa and to
use pyrolysis and GC/MS to identify the fingerprinted compounds. The number of individual
taxa that have been studied with respect to these methods is extremely small. In addition,
significant variability in fingerprint compounds exists between strains of a single taxon
(Sonesson et al., 1988).
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6. CONTROL OF BIOAEROSOL-INDUCED DISEASE
Very little actual research has gone into addressing the control of bioaerosol-induced.
disease, except in the area of immunization. A large amount of literature is available on
immunization to prevent infectious disease (Ruben, 1987) and immunotherapy for the control
of atopic disease (Ohman, 1989). These topics will not be discussed here. Other approaches
that are used, often in the absence of direct evidence of their efficacy, may be grouped as
follows (American Conference of Governmental and Industrial Hygienists, 1989b):
building design that provides for bioaerosol control,
. maintenance of indoor spaces so that contamination does not occur, and
remedial actions to control existing contamination.
*
6.1 BUILDING DESIGN
Buildings, including those for both public and private use, can be designed to prevent
penetration of outside aerosols to the indoor environment and to minimize the establishment
of indoor sources of biocontaminants. The purpose of the ASHRAE Standard 62-1989
(American Society of Heating, Refrigerating and Air-Conditioning Engineers, 1989) is "to
specify minimum ventilation rates and indoor air quality that will be acceptable to human
occupants and are intended to avoid adverse health effects". The standard, classifies
procedures for obtaining acceptable indoor air quality into two categories sine ventilation rate
procedure that specifies specific ventilation rates for given environments (Section 4.1) and
the indoor air quality procedure (Section 4.2) that requires control of known and specifiable
contaminants. Also specified are design criteria that are aimed at preventing penetration of
the outdoor aerosol and contamination of the ventilation system itself and controlling relative
humidity (see Chapter 5). Although supported by very little experimental or epidemiological
data, the standards, when applied to buildings without significant indoor sources, other than
human occupants, will go far in providing adequate air quality with respect to human source^
aerosols (Brundage et al., 1988) and problems related to relative humidity (Arundel et al.,
6-1
-------
1986; Carpenter etal.,' 1985). Unfortunately, the standards are rarely applied to domestic
buildings, and the use of .the "indoor air quality" procedures do not work for.bioaerosols
because there are no standards to use as guidelines. However, some control of residential air
quality can be obtained by the use of central or room air-conditioning (Spiegelman and
Friedman, 1968; Solomon et al.; 1980; Carpenter et al., 1985)., Air-conditioning allows
homes to be closed, preventing penetration of outdoor aerosols, and acts to reduce relative
> :. f
humidity.
6.2 BUILDING MAINTENANCE
Even if a building is properly designed, lack of adequate maintenance will inevitably
result in bioaerosol problems. Buildings must be maintained free of leaks and excessive
moisture. Filters cannot be allowed to build up a layer of organic material that will support
the growth of fungi or bacteria (B'urge and Garrison, 1989; Baxter, 1982). Air conditioners,
as well as controlling bioaerosols, may act as sources if not properly maintained (Banaszak
et al., 1970). Unfortunately, maintenance of home air-conditioners is rarely simple.
Humidifiers, whether centrally installed or console, are always contaminated with
microorganisms and often provide a built-in dissemination mechanism. A relatively large
amount of literature has been developed on the role of humidification in outbreaks of
infectious disease, HP, and sick building syndrome (Burge et al., 1987; Finnegan et al,,
1984; Kreiss and Hodgson, 1984; Kreiss, 1989). Less clear are methods for preventing
contamination and dissemination of organisms and antigens from these reservoirs.
Dust control measures may also help to prevent biological contamination of the indoor
environment. High-efficiency vacuuming of carpeting may help to prevent explosion of mite
populations (Walshaw and Evans,. 1986; Arlian et al., 1982). Limiting indoor food and
water sources,for cockroaches can prevent severe infestations hi some parts of the country.
6.3 REMEDIAL ACTIONS
Once contamination has occurred, several approaches may be taken to rid the
environment of the pollutant: air cleaning, air disinfection, and source control. Air cleaning
' - 6-2
-------
or particle removal is a function of the efficiency of the removal apparatus (e.g., filter,
electrostatic precipitator), the rate of flow through the device, and the strengths and rates of
emission of sources in the environment. To date, insufficient research has been conducted to
i '.;,..,,
allow a prediction of the effectiveness of air cleaning in the presence of active sources
(Nelson et al., 1988). However, it probably more efficiently removes small particles that
remain airborne for long periods of time. Also of concern is the downflovv from the cleaner,
which can disturb settled dust and entrained antigens and microorganisms.
The use of ultraviolet light has been studied as a means of removing infectious agents
»
from the indoor environment (Perkins et al., 1947; Rentschler and Nagy^.1942; Riley and
Kaufman, 1971; Wells et al., 1942). Ultraviolet radiation is dangerous and human contact
' "3 (
should be avoided. Also, ultraviolet lights only emit the appropriate wavelengths when
completely clean. Placing units in ductwork to kill recirculated organisms works only so
long as dust has not accumulated on the bulb surfaces.
Germicidal sprays have also been proposed for killing infectious organisms in air
(Kethley et al., 1956). These methods are not practical for occupied environments and all
residual disinfectant must be removed before occupancy is resumed. Biocides should never
be sprayed into ventilation systems in occupied buildings.
Source control involves removing the contaminated substrate from the environment,
removing the contamination, or killing organisms without removal. The first is usually the
most effective, although often not always practical. Immediate removal of soft materials that
have come hi contact with contaminated water is usually recommended because soft material
that is impregnated with fungal or bacterial growth is not retrievable. Removing living
antigen sources (househoWrpeis) is by far the best method for control ofl'&e contamination
they produce. Contamination can often be removed from hard surfaces. The
recommendation that smooth-surfaced flooring be used in bedrooms to prevent house dust
accumulation is a case in point. Dust can be removed from smooth floors by vacuuming or
wet mopping, whereas it inevitably accumulates in carpeting. Finally, biocides can be used
to kill organisms, although few biocides safe for use in occupied environments will prevent
recurrence of contamination (American Conference of Governmental and Industrial
Hygienists, 1989b). '
6-3
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