Chapter 14 - Principles of Disease and Epidemiology

Introduction
Disease- causing microorganisms are called pathogens.
Pathogenic microorganisms have special properties that allow them to invade the human body
or produce toxins.
When a microorganism overcomes the bodys defenses, a state of disease results.
Pathology, Infection, and Disease
Pathology is the scientific study of disease.
Pathology is concerned with the etiology (cause), pathogenesis (development), and effects of
disease.
Infection is the invasion and growth of pathogens in the body.
A host is an organism that shelters and supports the growth of pathogens.
Disease is an abnormal state in which part or all of the body is not properly adjusted or is
incapable of performing normal functions (loss of homeostasis).
Normal Microbiota
Animals, including humans, are usually germ-free in utero.
Microorganisms begin colonization in and on the surface of the body soon after birth.
Microorganisms that establish permanent colonies inside or on the body without producing
disease make up the normal microbiota (normal flora).
Transient microbiota are microbes that are present for various periods and then disappear.
Relationships Between the Normal Microbiota and the Host
The normal microbiota can prevent pathogens from causing an infection this phenomenon is
known as microbial antagonism.
Normal microbiota and the host exist in symbiosis (living together).
There are three types of symbiosis:

Commensalism (one organism benefits and the other is unaffected)

Mutualism (both organisms benefit)
Parasitism (one organism benefits and one is harmed)

Opportunistic Microorganisms

Opportunistic pathogens do not cause disease under normal conditions but cause disease
under special conditions.
Cooperation Among Microorganisms
In some situations, one microorganism makes it possible for another to cause a disease or
produce more severe symptoms.
The Etiology of Infectious Diseases
Kochs Postulates
Kochs postulates are criteria for establishing that specific microbes cause specific diseases.
Kochs postulates have the following requirements:

The same pathogen must be present in every case of the disease.

The pathogen must be isolated in pure culture.
The pathogen isolated from pure culture must cause the same disease in a
healthy, susceptible laboratory animal.
The pathogen must be re-isolated from the inoculated laboratory animal.

Exceptions to Kochs Postulates

Kochs postulates are modified to establish etiologies of diseases caused by viruses and
some bacteria which cannot be grown on artificial media.
Some diseases, such as tetanus, have unequivocal signs and symptoms.
Some diseases, such as pneumonia and nephritis, may be caused by a variety of microbes.
Some pathogens, such as S. pyogenes, cause several different diseases.
Certain pathogens, such as HIV, cause disease in humans only.
Classifying Infectious Diseases
A patient may exhibit symptoms (subjective changes in body functions) and signs (measurable
changes), which a physician uses to make a diagnosis.
A specific group of symptoms or signs that always accompanies a specific disease is
called a syndrome.
Communicable diseases are transmitted directly or indirectly from one host to another.
A contagious disease is one that is easily spread from one person to another.
Non-communicable diseases are caused by microorganisms that normally grow outside the
human body and are not transmitted from one host to another.
The Occurrence of a Disease
Disease occurrence is reported by incidence and prevalence.

Incidence Number of people contracting the disease in a given time period

Prevalence Number of total cases at a particular time regardless of when they

developed

Diseases are classified by frequency of occurrence:

Sporadic occurs occasionally (typhoid fever in the U.S.)

Endemic constantly present in a population (common cold)
Epidemic many cases in a given area in short period (influenza)
Pandemic a world wide epidemic (influenza occasionally, AIDS might be
considered pandemic)

The Severity or Duration of a Disease

The scope of a disease can be defined as:

Acute develops rapidly but lasts a short time (influenza)

Chronic develops more slowly, reactions to the disease are less severe, likely
to be continual or recurrent for long periods (infectious mononucleosis, TB,
hepatitis B)
Subacute intermediate between acute and chronic (sclerosing panencephalitis
a progressive/slow viral disease)
Latent the causative agent remains inactive for a time but then becomes active
to produce symptoms (shingles)

Herd immunity is the presence of immunity to a disease in most of the population.

The Extent of Host Involvement
A local infection affects a small area of the body; a systemic infection is spread throughout the
body via the circulatory system.
A secondary infection can occur after the host is weakened from a primary infection.
An inapparent, or subclinical, infection does not cause any signs of disease in the host.
Patterns of Disease
Predisposing Factors
A predisposing factor is one that makes the body more susceptible to disease or alters the
course of a disease.
Examples include gender, climate, age, fatigue, and inadequate nutrition.
The Development Of Disease

Incubation period - the time interval between the initial infection and the first appearance of
signs and symptoms.
Prodromal period - characterized by the appearance of the first mild signs and symptoms.

Period of illness - the disease is at its height, and all disease signs and symptoms are
apparent.

Death may occur during the period of illness.

Crisis is the phase of fever characterized by vasodilation and sweating (the body
is trying to return to normal temperature, the fever is breaking).

Period of decline - the signs and symptoms subside.

Period of convalescence - the body returns to its prediseased state, and health is restored.

The Spread of Infection

Reservoirs of Infection
A continual source of infection is called a reservoir of infection.
Living Reservoirs
Human reservoirs of infection:
People who have a disease..
People who are carriers of pathogenic microorganisms.
Zoonoses are diseases that affect wild and domestic animals and can be transmitted to
humans.
Nonliving Reservoirs
Soil (fungi, C. botulinum, C. tetani)
Water (V. cholerae, S. typhi, protozoans, algae)
Improperly prepared or stored foods (trichinosis and salmonellosis)
The Transmission Of Disease
Contact Transmission

Direct contact (person-to-person) involves close physical contact between the source
of the disease and a susceptible host.
Indirect contact involves transmission by fomites (inanimate objects).
Droplet transmission is transmission via droplet nuclei (mucus droplets) in coughing or
sneezing, laughing or talking.

Vehicle Transmission
Common vehicle transmission is transmission by a medium such as water, food or air.

Waterborne
Foodborne

Airborne - pathogens are carried on droplet nuclei in dust for a distance greater than
1 meter.

Vector Transmission
Arthropod vectors carry pathogens from one host to another by both mechanical and biological
transmission.

Mechanical transmission is the passive transport of a pathogen on a vector 's feet or

other body parts.
Biological transmission involves reproduction of the pathogen in the vector and
transmission in saliva or feces.

Nosocomial (Hospital-Acquired) Infections

A nosocomial infection is any infection that is acquired during the course of stay in a hospital,
nursing home, or other health care facility.
About 5-15% of all hospitalized patients acquire nosocomial infections.
Three factors contribute to nosocomial infections:

Microorganisms in the hospital

A compromised host
The chain of transmission

Microorganisms In The Hospital

Certain normal microbiota are often responsible for nosocomial infection when they are
introduced into the body through such medical procedures as surgery and catheterization.
Opportunistic, drug-resistant gram-negative bacteria are the most frequent causes of
nosocomial infections.
The Compromised Host
Patients with burns, surgical wounds, and suppressed immune systems are the most
susceptible to nosocomial infections.
The Chain Of Transmission
Nosocomial infections are transmitted by direct contact between staff members and patients
and between patients.
Fomites such as catheters, syringes, and respiratory devices can transmit nosocomial
infections.
The Control Of Nosocomial Infections
Aseptic techniques can prevent nosocomial infections.

Hospital infection control staff members are responsible for overseeing the proper cleaning,
storage, and handling of equipment and supplies.
Emerging Infections Diseases
New diseases and diseases with increasing incidences are called emerging infectious
diseases (EIDs).
EIDs can result from the use of antibiotics and pesticides, climatic changes, travel, the lack of
vaccination, and insufficient case reporting.
The CDC, NIH, and WHO are responsible for surveillance and responses to emerging
infectious diseases.
Epidemiology
The science of epidemiology is the study of the transmission, incidence, and frequency of
disease.
Case Reporting
Case reporting provides data on incidence and prevalence to local, state, and national health
officials.
Establishes the chain of transmission.
The Centers for Disease Control and Prevention (CDC), a branch of the U.S. Public Health
Service, is the main source of epidemiologic information in the United States.
The CDC publishes the Morbidity and Mortality Weekly Report to provide information on
morbidity (incidence) and deaths (mortality).
Morbidity rate is the number of people affected by a disease in a given period of time
(incidence) in relation to the total population.
Mortality rate is the number of deaths resulting from a disease in a population in a given
period of time in relation to the total population.
Notifiable diseases - cases are required by law to be reported to the U.S. Public Health Service
Chapter 15 - Microbial Mechanisms of Pathogenicity
Introduction
Pathogenicity is the ability of a pathogen to produce a disease by overcoming the defenses of
the host.
Virulence is the degree of pathogenicity.
How Microorganisms Enter A Host
Portals of Entry

The specific route by which a particular pathogen gains access to the body is called a portal of
entry.
Mucous membranes
Respiratory tract
Microorganisms that are inhaled with droplets of moisture and dust particles gain
access to the respiratory tract.
The respiratory tract is the most common portal of entry.
Genitourinary tract
Microorganisms that gain access via the genitourinary tract can enter the body
through mucous membranes.
Gastrointestinal tract
Microorganisms enter the gastrointestinal tract via food, water, and contaminated
fingers.
Conjunctiva
Skin
Most microorganisms cannot penetrate intact skin; they enter hair follicles and sweat
ducts.
Some fungi infect the skin itself.
Parenteral
Some microorganisms can gain access to tissues by inoculation through the skin and
mucous membranes in bites, injections, and other wounds.
The Preferred Portal of Entry
Many microorganisms can cause infections only when they gain access through their specific
portal of entry.
Numbers of Invading Microbes
Virulence can be expressed as LD50 (lethal dose for 50% of the inoculated hosts) or ID 50
(infectious dose for 50% of the inoculated hosts).
Lower LD50 or ID50 = more virulent organism, stronger toxin.
Adherence

Surface projections on a pathogen called adhesins (ligands) adhere to complementary

receptors on the host cells.
Ligands can be glycoproteins or lipoproteins and are frequently associated with
fimbriae.
Mannose is the most common receptor on host cells.
Biofilms are communities of microorganisms which, together with their extracellular products,
attach to living or nonliving surfaces.
Usually form on surfaces that are moist and contain organic substances.
Bacteria usually attach first, multiply and secrete glycocalyx, allows attachment of
bacteria to each other and to the surface.
May be several layers thick .
May contain different types of microorganisms.
Resist disinfectants and antibiotics.
Biofilms may form on teeth, catheters, contact lenses, etc. - estimated that 65% of human
bacterial infections involve biofilms.
Examples: Dental plaque, soap scum, algae on swimming pool walls
How Bacterial Pathogens Penetrate Host Defenses
Capsules
Some pathogens have capsules that prevent them from being phagocytized.
Components of the Cell Wall
M protein of Streptococcus pyogenes.
Heat- and acid-resistant protein in the cell wall and fimbriae that facilitate
adherence to epithelial cells and prevent phagocytosis by leukocytes.
Opa - outer membrane protein Neisseria gonorrhoea uses with fimbriae to attach to host
cells and gain entry (live inside epithelial cells and leukocytes).
Waxes in cell wall of Mycobacterium tuberculosis.
Some microbes can reproduce inside phagocytes.
Enzymes
Leukocidins destroy neutrophils and macrophages.
Hemolysins lyse red blood cells.

Local infections can be protected in a fibrin clot caused by the bacterial enzyme
coagulase.
Bacteria can spread from a focal infection by means of kinases (which destroy blood
clots), hyaluronidase (which destroys a mucopolysaccharide that holds cells together),
and collagenase (which hydrolyzes connective tissue collagen).
IgA proteases destroy IgA antibodies.
Antigenic Variation
Some organisms change their antigens, either by mutation or by changing expression of
the gene that codes for the antigen, to evade host immune responses.
Penetration into the Host Cell Cytoskeleton
Salmonella produce invasins, proteins that cause the actin of the host cells
cytoskeleton to form a "basket" (membrane ruffling) to carry the bacteria into the cell.
How Bacterial Pathogens Damage Host Cells
Bacteria damage host cells four ways:

Using the host's nutrients

Direct damage
Toxins
Hypersensitivity reactions

Using the Host's Nutrients

Bacteria take iron from the host's iron transport proteins by secreting siderophores,
which bind iron even more tightly.
Direct Damage
Host cells can be destroyed when pathogens metabolize and multiply inside the host
cell.
The Production of Toxins
Poisonous substances produced by microorganisms are called toxins; toxemia refers to
the presence of toxins in the blood. The ability to produce toxins is called toxigenicity.
Exotoxins are produced by bacteria (mostly gram-positive) and released into the
surrounding medium. Exotoxins, not the bacteria, produce the disease symptoms.
Antibodies produced against exotoxins are called antitoxins.
May be named according to:

The type of cells they attack (neurotoxins, enterotoxins, cytotoxins [wide variety
of cells, includes cardiotoxins, hepatotoxins, and leukotoxins]),
The diseases they are associated with (diptheria toxin, tetanus toxin)
The bacterium that produces them (botulinum toxin, Vibrio enterotoxin)

There are three basic types of exotoxin based on structure and function:

A-B toxins (B subunit binds to the cell, toxin is taken into the cell, the A subunit

enzymatically kills the cell)

Membrane disrupting toxins (disrupt host cell plasma membranes)
Superantigens (provoke intense immune responses)

Endotoxins are lipopolysaccharides (LPS), the lipid A component of the wall of gram-negative
bacteria.
Bacterial cell death, antibiotics, and antibodies may cause the release of endotoxins.
Endotoxins cause fever (by inducing the release of interleukin-1) and shock (because of
a TNF-induced decrease in blood pressure).
TNF causes edema by damaging capillaries.
LPS stimulates release of NO from macrophages, which causes vasodilation.
Endotoxins allow bacteria to cross the blood-brain barrier.
Property
Type molecule
Effects
Stability
Toxicity
Lethal Dose
Antigenicity

Exotoxin

Endotoxin
Proteins
Lipid; The lipid A portion of
lipopolysaccharide found in the
cell wall of gram - bacteria
Specific to cells or functions,
General effects: fever, may
doesnt cause fever: cytotoxins, cause shock. All endotoxins
neurotoxins, enterotoxins
have the same effects.
Usually easily destroyed by heat Very stable, can withstand
(except staphylococcal
autoclaving (121C for 1 hour)
enterotoxin)
High
Low
Low
High
Can be used as a toxoid,
Not a good toxoid, not easily
produces antitoxins when used
neutralized by antitoxins.
as a vaccine.

Plasmids, Lysogeney, and Pathogenicity

Plasmids may carry genes for antibiotic resistance, toxins, capsules and fimbriae.
Lysogenic conversion can result in bacteria with virulence factors, such as toxins or capsules.

Portals of Exit
Just as pathogens have preferred portals of entry they also have definite portals of exit.

The respiratory tract via coughing or sneezing.

The gastrointestinal tract via saliva or feces.
The urogenital tract via secretion from the vagina or penis.
Wounds
Arthropods
Syringes
Chapter 16 - Nonspecific Defenses of the Host

Introduction
The ability to ward off disease through body defenses is called resistance.
Lack of resistance is called susceptibility.
Nonspecific resistance refers to all body defenses that protect the body against any kind of pathogen.
First Line of Defense

Skin
Mucous membranes

Second line of defense

Phagocytosis
Inflammation
Complement
Interferons
Fever

Third line of defense

Immunity
Specific resistance (immunity) refers to defenses (antibodies) against specific microorganisms.

Phagocytosis
Phagocytosis is the ingestion of microorganisms or particulate matter by a cell.
Phagocytes are activated by bacterial components (for example, lipid A) and cytokines.
Actions of Phagocytic Cells
Among the granulocytes, neutrophils are the most important phagocytes.
Enlarged monocytes become wandering macrophages and fixed macrophages.

Fixed macrophages are located in selected tissues and are part of the mononuclear
phagocytic system.
Granulocytes predominate during the early stages of infection, whereas macrophages predominate
as the infection subsides.
The Mechanism of Phagocytosis
Chemotaxis is the process by which phagocytes are attracted to microorganisms.
The phagocyte then adheres to the microbial cells; macrophages have cell-surface receptors that
recognize certain molecules on the surface of various pathogens, including those containing
mannose. When a pathogen displays a cell surface polysaccharide containing mannose it is bound by
these phagocytosis receptors (more on this concept later).
Adherence may be facilitated by opsonization coating the microbe with serum proteins, like
complement or antibodies.
Pseudopods of phagocytes engulf the microorganism and enclose it in a phagocytic vesicle to
complete ingestion.
Many phagocytized microorganisms are killed by lysosomal enzymes and oxidizing agents.
Microbial Evasion of Phagocytosis
Some microbes are not killed by phagocytes and can even reproduce in phagocytes.
Evasion mechanisms include M protein, capsules, leukocidins, membrane attack complexes, and
prevention of phagolysosome formation.
Inflammation
Inflammation is a bodily response to cell damage.
The four cardinal signs of inflammation, as described by the Roman physician and science writer
Celsus, are:

Rubor - redness
Tumor - swelling
Calor - heat
Dolor pain
(The fifth sign, which is sometimes present, is loss of function, or functio laesa - this was
originally described and added to the four signs described by Celsus by another Roman
physician and science writer, Galen, but was popularized in the 1800s by Rudolph Virchow, the
"Father of Modern Pathology". )

Functions:

To destroy and remove pathogens and debris.

To confine pathogens; prevent spread of infection.

To repair or replace damaged tissue (sets stage for wound repair).

Vasodilation and Increased Permeability of Blood Vessels

Injured tissue cells, neutrophils, macrophages, dendritic cells, mucosal epithelial cells, endothelial
cells, basophils, and lymphocytes all release inflammatory mediators.
The release of histamine, kinins, and prostaglandins causes vasodilation and increased
permeability of blood vessels, sensitization and/or stimulation of pain receptors, and activation
of clotting factors.
The release of cytokines like tumor necrosis factor-alpha (TNF-), interleukin-1 (IL-1), and
chemokines, act as chemoattractants for white blood cells and promote inflammation at the
site of interaction.
Blood clots can form around an abscess to prevent dissemination of the infection.
Epithelial mucosal cells increase their release of - defensins (broad spectrum antimicrobial
proteins) when the epithelial barrier has been breached and the underlying connective tissue is
inflamed.
Phagocyte Migration and Phagocytosis
Phagocytes have the ability to stick to the lining of the blood vessels (margination).
They also have the ability to squeeze through blood vessels (emigration).
PMNs show up first and release ROI (reactive oxygen intermediates), like superoxide anions,
hydroxyl ions, hydrogen peroxide, and the enzyme myeloperoxidase, which converts hydrogen
peroxide to hypochlorous acid (HOCl, which dissociates to H + and OCl-, the hypocholorite anion,
basically bleach). PMNs also release defensins and are phagocytic.
Pus is the accumulation of damaged tissue and dead microbes, granulocytes, and some
macrophages. Generally macrophages show up late to clean up the cellular debris (debride the
wound) and set the stage for wound healing.
Tissue Repair
A tissue is repaired when the stroma (supporting tissue) or parenchyma (functioning tissue) produces
new cells.
Synthesis of parenchymal tissue to repair the wound is healing by 1 st intent and produces functional
repaired tissue
Stromal repair by fibroblasts produces scar tissue (healing by 2 nd intent).
Fever

Fever is an abnormally high body temperature produced in response to a bacterial or viral infection.
Bacterial endotoxins and interleukin-1 can induce fever.
A chill indicates a rising body temperature; crisis (sweating) indicates that the bodys temperature is
falling.
The Complement System
The complement system consists of a group of serum proteins that activate one another to destroy
invading microorganisms.
There are three pathways to complement activation but they all end up forming a C3 convertase
complex which activates C3 and results in three things:

Inflammation
Opsonization
Cell lysis

C3 is cleaved to C3a and C3b

C3a stimulates inflammation
C3b opsonizes cells and cleaves C5 to produce C5a and C5b
C5a stimulates inflammation
C5b gets C6, C7, C8, and C9 to associate to form the MAC, which causes cell lysis.
Activation of Complement
Classical Pathway
C1 binds to antigen antibody complexes. (Antibody binding to antigen is called "antigen
fixation"; when an antigen-antibody complex binds C1 this is called "complement fixation")
C2 and C4 associate and this acts as C3 convertase.
Alternative Pathway
Factor B, factor D, factor P, and C3b bind to certain cell wall polysaccharides (i.e.
peptidoglycan) to activate C3b.
C3 undergoes autolysis to provide C3b, which is very unstable and has a short half-life.
Factor B stabilizes the C3b.
Factor D cleaves factor B to produce a C3b-factorBb molecule; this acts as C3 convertase.
Properdin, Factor P, stabilizes the C3b-factorBb C3 convertase.

The Lectin Pathway

Phagocytosis by macrophages induces release of chemicals that stimulate the liver to produce
carbohydrate binding proteins (lectins).
One such lectin, mannose-binding lectin (MBL) binds to mannose on bacterial cells walls and
on some viruses.
MBL opsonizes and activates C2 and C4 to activate C3.
Interferons
Interferons (IFNs) are antiviral proteins produced in response to viral infection.
There are three types of human interferon: -IFN, -IFN, and -IFN. Recombinant interferons
have been produced.
The mode of action of -IFN and -IFN is to induce uninfected cells to produce antiviral protein
(AVPs) that prevent viral replication.
Interferons are host-cell-specific but not virus-specific.
-IFN activates neutrophils and macrophages to kill bacteria.
-IFN also activates Th1 cells, which stimulate cell mediated reactions.
Lack of -IFN results in activation of Th2 cells, which are humoral mediators
Very high levels of -IFN stimulates NK cells and CTLs.
Transferrins
Iron binding proteins found in blood, milk, saliva, and tears.
Reduce microbial growth by denying microorganisms access to iron.
Chapter 17 - Adaptive Immunity: Specific Defenses of the Host

Chapter 18 Applications of Immunology

Vaccines
A vaccine is a substance that when injected causes the production of specific antibodies or
activated T-cells.
Herd immunity results when most of a population is immune to a disease. The purpose of
vaccination is to establish herd immunity when it does not already exist.

Types of vaccines:
Attenuated whole-agent vaccines consist of attenuated (weakened) microorganisms:
attenuated virus vaccines generally provide lifelong immunity.
Inactivated whole-agent vaccines consist of killed bacteria or viruses.
Toxoids are inactivated toxins.
Subunit vaccines consist of antigenic fragments of a microorganism; these include
recombinant vaccines and acellular vaccines.
Conjugated vaccines combine the desired antigen with a protein that boosts the
immune response.
Nucleic acid vaccines, or DNA vaccines, are being developed. These cause the
recipient to make the antigenic protein associated with class I MHC (HLA).
Adjuvants improve the effectiveness of some antigens.
Viruses for vaccines may be grown in animals, cell cultures, or chick embryos.
Vaccines are the safest and most effective means of controlling infectious diseases.
Complications of Vaccines
Problems in administration
Administration to pregnant women
Infection
Local reactions at the injection site
Fever
Allergies
Development of disease
Mutation to a virulent strain
Diagnostic Immunology
Serologic tests to determine the presence of antibodies or antigens in a patient are based on the fact
that antibodies bind to specific antigens.
These tests require 2 things:
A source of specific antibodies
A way to visualize the antigen-antibody interaction
Monoclonal Antibodies
Hybridomas - produced by the fusion of malignant cells and plasma cells. The resulting population of
cells is immortal and able to produce large amounts of a specific antibody.
Uses:
Serologic identification
Prevention of tissue rejections
Cancer research
Immunotoxins can be produced by combining monoclonal antibody with toxin

o
o

Immunotoxins are targeted to react with specific antigens

Precipitation Reactions
The interaction of soluble antigens with IgG or IgM antibodies leads to precipitation reactions.
Precipitation reactions depend on the formation of lattices and occur best when antigen and antibody
are present in optimal proportions.

The precipitin ring test is performed in a small tube.

Antibodies from the bottom of the tube and antigens from the top of the tube diffuse
toward each other and when the optimal antigen-antibody ratio is reached a visible ring
(precipitate) appears in the tube.
Immunodiffusion procedures are precipitation reactions carried out in an agar gel medium.
Antibody and antigen are loaded in different wells and diffuse through the medium.
When the optimal antigen-antibody ratio is reached a visible band appears in the gel.

Agglutination Reactions
The interaction of particulate antigens (cells that carry antigens) with antibodies leads to agglutination
reactions.
Diseases can be diagnosed by a rising titer (antibody concentration in serum) or seroconversion
(from no antibodies to the presence of antibodies).
Direct agglutination reactions test patient serum against large, cellular antigens to screen for the
presence of antibodies. Antibodies cause agglutination of the cells.
Indirect agglutination reactions can be used to test patient serum for the presence of antibodies
against soluble antigens.
Serum is mixed with latex spheres with the soluble antigens attached. Antibodies will then cause
visible agglutination of the latex spheres with the soluble antigens attached.
Alternatively, antibodies may be attached to the latex spheres to test for the presence of soluble
antigens in patient serum.
Hemagglutination reactions involve agglutination reactions using red blood cells.
Neutralization Reactions
In a toxin neutralization test, the presence of antibodies against a toxin can be detected by the
antibodies ability to prevent toxic effects in cells.
In a virus neutralization test, the presence of antibodies against a virus can be detected by the
antibodies ability to prevent cytopathic effects of viruses in cell cultures.
Complement-Fixation Reactions
There are two steps, the complement fixation step and the indicator step.
Complement Fixation Step
Add antigen and complement to serum. If the serum contains antibodies against the antigen they will
bind to the antigen and fix the complement.
This ties up all the free complement so it can't participate in the next step, the indicator step.

Indicator Step
Add sheep red blood cells and anti-sheep red blood cell antibodies to the serum. Antibodies to the
sheep red blood cells bind and can fix complement, if any is available.
If complement is available it will be fixed by the sheep red blood cell antigen-antibody complex and
the sheep red blood cells will be lysed. This indicates that the serum did not contain antibodies
against the antigen added in the complement fixation step and complement remained free.
If no complement is available the sheep red blood cells will not be lysed. This indicates there were
antibodies against the antigen added in the complement fixation step and all the complement was tied
up when it was fixed by the original antigen-antibody complex.
Fluorescent-Antibody Techniques
Direct fluorescent-antibody tests are used to identify specific microorganisms/antigens in patient
samples.
Antibodies directed against antigens on the surface of a specific microorganism are labeled
with fluorescent dye.
Fluorescent antibodies are incubated with the patients sample and antigen-specific binding
allowed to occur.
The sample is viewed with a fluorescence microscope or plate reader or fluorescenceactivated flow cytometer.
Indirect fluorescent-antibody tests are used to demonstrate the presence of antibody in serum.
Antigen or the microorganism itself is incubated with the patient's serum and any antibodies
against the antigen or organism that are present in the patients serum allowed to bind.
Fluorescent anti-human immunoglobulin antibodies are then added and will bind to any patient
antibodies present. (Inject human immunoglobulins into another species and it will produce
anti-human immunoglobulin antibodies).
The sample is viewed with a fluorescence microscope or plate reader or fluorescenceactivated flow cytometer.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA techniques use antibodies linked to an enzyme, such as horseradish peroxidase or alkaline
phosphatase.
Antigen antibody reactions are detected by enzyme activity. The substrate for the enzyme is
converted to a chromogenic product in the indicator step.
The direct ELISA is used to detect specific antigens in a patient's serum.
Coat the bottom of a test well with an antibody against the antigen. Then add patient serum. If
the antigen is present in the patient's serum it will bind to the antibody that is attached to the
bottom of the well (the capture antibody).

Next add enzyme-linked antibody, which is also specific for the antigen its the same antibody
that coated the bottom of the well except it has an enzyme linked to it. If the antigen is present
you'll end up with an antibody-antigen-enzyme-linked antibody sandwich.
Add substrate. If there is any enzyme-linked antibody present (and it should only be there if it
is bound to antigen) a product will be formed that causes the color change.
The indirect ELISA is used to detect antibodies in patient serum against a specific antigen.
Coat the bottom of the well with the antigen that the antibody would be specific for. Then add
patient serum. Any antibodies specific for the antigen will bind.
Next add enzyme-linked anti-human immunoglobulin antibody.
If the patient's serum had antibodies against the antigen coatin the bottom of the well you'll end
up with an antigen-antibody-enzyme-linked anti-human immunoglobulin antibody sandwich.
Add substrate and look for the color change just like in the direct ELISA.
Chapter 19 - Disorders Associated With the Immune System
Hypersensitivities are altered immune reactions (in response to an antigen) leading to tissue damage,
pretty much the same thing as an allergy.
An allergen is an antigen that stimulates a hypersensitivity response.
Immediate hypersensitivities are based on humoral immunity and include Types I, II, and III.
Delayed is based on cell-mediated immunity, Type IV.
Type I (Anaphylaxis) Reactions
Anaphylaxis reactions occur when antigen stimulates IgE production. (This is sensitization.)
IgE antibodies bind to basophils and/or mast cells by their stem region, leaving the antigen
binding sites free.
When adjacent IgE antibodies bound to basophils and/or mast cells are cross-linked by binding
to antigen it results in the release of mediators (histamine, leukotrienes and prostaglandins)
that cause inflammation.
There are two basic kinds of reactions: systemic anaphylaxis and localized reactions.
Anaphylactic reactions can be prevented by determination of the specific allergens that a
patient is sensitive to and injecting small amounts of the allergens over an extended period of
time (desensitization). This causes the production of blocking antibodies, which are IgG.
Type II (Cytotoxic) Reactions
The antibodies are directed toward cellular antigens on foreign cells or foreign antigens on
host cells.
The antigen-antibody complexes cause complement fixation resulting in cell lysis and
phagocytosis. Examples include incompatible blood transfusions, Rh incompatibility, and druginduced cytotoxic reactions.

Type III (Immune Complex) Reactions

Antigens involved are not part of host cells but soluble antigens.
The antigens are bound by IgM or IgG antibodies and the antigen-antibody complexes
precipitate and lodge in basement membranes of blood vessels.
Complement fixation leads to inflammation and cell lysis. An examples is glomerulonephritis.
Type IV (Cell Mediated) Reactions
Delayed-type hypersensitivity (TDTH) T-cells are involved.
Sensitized T-cells secrete lymphokines in response to antigen. Lymphokines attract
macrophages and initiate tissue damage. Examples include the tuberculin skin test and allergic
contact dermatitis.
Autoimmune Diseases
Due to loss of self-tolerance (tolerance is the ability to recognize self proteins as self and not
as foreign).
Antibodies to infectious agents may cross react with self-proteins on host cells and cause
damage to the cells. Examples include autoimmune and rheumatic fever.
Antibodies may bind to host cell surface antigens without cell destruction. Examples include
Graves disease and myasthenia gravis.
Antibodies may bind to soluble host antigens and the antigen-antibody complexes precipitate
and fix complement. Examples include systemic lupus erythematosus, and rheumatoid
arthritis.
Cell mediated autoimmunity involves activation of cytotoxic T-cells by infectious agents
followed by cross-reactivity with normal host cells. Examples include multiple sclerosis,
Hashimotos autoimmune thyroiditis, and insulin-dependent diabetes mellitus.
Immune Deficiencies
Immunodeficiencies may be congenital or acquired.
Congenial deficiencies are due to defective or absent genes.
Acquired immune deficiencies can caused by drugs, cancers, and infectious diseases.
Acquired Immunodeficiency Syndrome (AIDS)
AIDS is the final stage of HIV infections.
HIV is a retrovirus with a phospholipid envelope with gp 120 spikes which attach to CD4
receptors and coreceptors on host cells (helper T cells, macrophages, and dendritic cells).
HIV infection is categorized by symptoms:
Category A asymptomatic
Category B selected symptoms
Category C AIDS indicator conditions, reported as AIDS

Also categorized by CD4 T cell numbers: below 200/mm3 is reported as AIDS (true for
Category A and B also).
Progression from HIV infection to AIDS takes about 10 years.
Transmission is by sexual contact, breast milk, contaminated needles, transplacental infection,
artificial insemination, and blood transfusion although blood transfusions are not a likely source
of infection in developed countries.
In the U.S., Canada, western Europe, Australia, northern Africa, and parts of South America
transmission has been by injecting drug use (IDU) and male-to-male sexual contact.
Heterosexual transmission is increasing.
In sub-Saharan Africa transmission is primarily heterosexual contact.
In Eastern Europe and Asia transmission is by IDU and heterosexual contact.
Worldwide the primary means of transmission is through unprotected (heterosexual)
sex.
Chapter 20 - Antimicrobial Drugs
Antimicrobial drugs should:
1. Have selective toxicity that is, should be toxic to the microbe not the host
2. Not provoke hypersensitivity
3. Be soluble in body fluids so that they can get into the areas where the infection exists
4. Should be cleared from the blood fast enough that toxic situations do not occur, but not so
fast that therapeutic doses cannot be reached.
5. Have a long shelf life this makes them less expensive, more readily available and
available for use in rural areas.
6. Not provoke resistance
Paul Ehrlich developed the concept of chemotherapy to treat microbial diseases: he developed
salvarsan to treat syphilis in 1910.

Alexander Fleming discovered the first antibiotic, penicillin, in 1928.

The Spectrum of Antimicrobial Activity
Antibacterial drugs affect many targets in a prokaryotic cell.
Fungal, protozoan, and helminthic infections are more difficult to treat without harming the host
because these organisms have eukaryotic cells.

Narrow spectrum drugs affect only a select group of microbesgram positive cells for example;
broad-spectrum drugs affect a wider range of different types of organisms.
The Action of Antimicrobial Drugs
Inhibit cell wall synthesis
Inhibit protein synthesis
Cause injury to plasma membranes
Inhibit nucleic acid synthesis
Inhibit enzyme activity
Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis
Penicillin
Penicillin inhibits cell wall synthesis in bacteria.
This of course requires that the bacteria are actively growing.
Since human cells do not have peptidoglycan cell walls penicillin has low toxicity; the primary
concern is for allergy, which only occurs in a low percentage of the population, making penicillin
the antibiotic with the fewest side effects.
All penicillins contain a B-lactam ring.
Natural Penicillins
Natural penicillins (Penicillin G, Penicillin V) produced by Penicillium are effective against grampositive cocci and spirochetes.
Natural penicillins have a narrow spectrum activity and are susceptible to penicillinases (or Blactimases) - bacterial enzymes that destroy natural penicillins.

Semisynthetic Penicillins
Semisynthetic penicillins are made in the laboratory by adding different side chains onto the B-lactam
ring after it is synthesized by a fungus.
Semisynthetic penicillins (oxacillin, ampicillin, amoxicillin, aztreonam, imipenem) are resistant to
penicillinases and have a broader spectrum of activity than natural penicillins.
The first penicillinase-resistant semisynthetic penicillin was methicillin. We've got so much methicillin
resistance that methicillin use has been discontinued in the U.S.
Vancomycin inhibits cell wall synthesis and may be used to kill penicillinase-producing staphylococci.
Used primarily against methicillin-resistant S. aureus.
Very toxic
Streptogramins are bactericidal agents that inhibit protein synthesis and may be used to kill
vancomycin-resistant bacteria.
Syncercid - combination of quinupristin and dalfopristin, blocks protein synthesis.

Effective against a broad range of gram-positive organisms but expensive and has a lot of side
effects.
Useful for treating VRSA but not Enterococcus faecalis (which is likely to be VRE, especially in
clinical settings).
Oxazolidinones are also useful against vancomycin resistance, especially enterococci that are
resistant to syncercid.
Zyvox - totally synthetic, may slow development of of resistance. There are some toxicity
issues and the course of treatment is 28 days.
Daptomycin, a new drug (the first lipopeptide to be released) produced by Streptomyces, binds to
bacterial cell membranes and causes rapid depolarization, the depolarized cell can't synthesize
nucleic acids and proteins and dies.
Effective only against gram positive organisms because it can't penetrate the outer membrane
of gram-negative organisms.
More rapidly bactericidal than vancomycin against Staphylococcus aureus and Enterococcus
(including MRSA and VRE).
Daptomycin has no identifiable transferable mechanism of resistance, however you may see
the statement "resistance has been seen" - 2 isolates out of more than 1000 courses of
therapy in clinical trials (1 was S. aureus and the other was E. faecalis)

Cephalosporins
Cephalosporins inhibit cell wall synthesis like penicillins and have more activity against gramnegative organisms.
They are used against penicillin resistant strains but are susceptible to a different class of Blactamases and also are contraindicated in people with penicillin allergy.
Antimycobacterial Antibiotics
Isoniazid (INH) inhibits mycolic acid synthesis in mycobacteria and is used to treat
tuberculosis. INH is administered with rifampin or ethambutol to avoid resistance.
Tetracyclines
Tetracyclines are said to have the broadest spectrum of all antibiotics; effective against both
gram-positive and gram-negative bacteria as well as against rickettsias, mycoplasmas and
chlamydias. Often used to treat urinary tract infections.
Can suppress the normal flora leading to superinfections of Candida albicans.
May cause discoloration of the teeth in children and liver damage in pregnant women.
Semisynthetic tetracylines (doxycycline and minocycline) have longer retention in the body
than the natural tetracylcines.

Macrolides (Example: Erythromycin) Spectrum similar to that of penicillin G; good alternative to

penicillin; Drug of choice for legionellosis and mycoplasmal pneumonia.
Newer macrolides include azithromycin and clarithromycin (Biaxin) - broader spectrum and
penetrate tissues better (good against Chlamydia).
Even newer are ketolides, developed to deal with resistance. Example: telithromycin (Ketek)
Quinolones are a group of antibiotics that inhibits DNA synthesis by effecting DNA gyrase, which is
needed for replication.
Use is limited to urinary tract infections.
Fluoroquinolones - have a broad spectrum of activity, penetrate tissues well, and are safe for
adults.
They are not recommended for children, adolescents, and pregnant women because they may
adversely affect cartilage development.
Norfloxacin, ciprofloxacin (Cipro), and levofloxacin (Levoquin) are the most commonly used.
Sulfonamides (sulfa drugs) are bacteriostatic antibiotics.
The most widely used sulfa drug is trimethoprim-sulfamethoxazole (Cotrimoxazole, Septra),
which interferes with folic acid metabolism.
It is very effective in penetrating brain tissue and cerebrospinal fluid.
Tests To Guide Chemotherapy
These tests are used to determine which chemotherapeutic agent is most likely to combat a
specific pathogen.
These tests are used when susceptibility cannot be predicted or when drug resistance arises.
The Diffusion Methods
In the Kirby-Bauer test a bacterial culture is inoculated on an agar medium, and filter paper
disks impregnated with chemotherapeutic agents are overlaid on the culture.
After incubation, the absence of microbial growth around a disk is called a zone of inhibition.
The diameter of the zone of inhibition, when compared with a standardized reference table, is
used to determine whether the organism is sensitive, intermediate, or resistant to the drug.
MIC (minimal inhibitory concentration) is the lowest concentration of drug capable of
preventing microbial growth; MIC can be estimated using the E test.
A plastic coated strip contains a gradient of antibiotic concentrations and the minimal inhibitory
concentration is read from a scale printed on the strip.
Broth DilutionTests

In a broth dilution test, the microorganism is grown in a liquid media containing different
concentrations of a chemotherapeutic agent.
The lowest concentration of a chemotherapeutic agent that kills bacteria is called the
minimum bactericidal concentration (MBC).