Beruflich Dokumente
Kultur Dokumente
The first two lectures of the virology section deal with basic concepts while the
subsequent three lectures deal with individual virus families and their medical
significanse and importance.
The lecture on mycology presents succinct summary of the facts about medically
important fungi.
The first lecture of the oral microbiology section deal with development of the oral
microflora while subsequent two lectures deal with the dental caries and the
periodontal diseases.
into genera and species. This was the beginning of the taxonomic classification of
microbes.
In 1840, the German pathologist Friedrich Henle proposed criteria for proving that
microorganisms were responsible for causing human disease (the “germ theory” of
disease). Koch and Pasteur confirmed this theory in the 1870s and 1880s with a series
of elegant experiments proving that microorganisms were responsible for causing
anthrax, rabies, plague, cholera, and tuberculois.
Other brilliant scientists went on to prove that a diverse collection of microbes was
responsible for causing human disease.
The era of chemotherapy was begun in 1910 when the German chemist Paul
Ehrlich discovered the first antibacterial agent, a compound effective against the
spirochete that causes syphilis. This was followed by Alexander Fleming’s discovery
of penicillin in 1928, Gerhard Domagk’s discovery of sulfanamide in 1935, and
Selman Waksman’s discovery of streptomycin in 1943.
In 1946, the American microbiologist John Enders was the first to cultivate viruses
in cell cultures, leading the way to the largescale production of virus cultures for
vaccine development. Thousands of scientists have followed thses pioneers, each
building on the foundation established by his or her predecessors, and each adding an
observation that expanded our understanding of microbes and their role in disease.
Microorganisms
Viruses
Viruses are the smallest infectious particles, ranging in diameter from 18 to almost
300 nm (particles less than 200 nm cannot be seen with a light microscope). More
than 40 genera of viruses have been implicated in human disease, and certainly, more
will be discovered each year. Viruses consist of either DNA or RNA (but not both)
and protein required for replication and pathogenesis. These components are then
enclosed in a protein coat with or without a lipid membrane coat. These organisms
are true parasites, requiring host cells for replication.
Bacteria
Bacteria are relatively simple in structure. They are procaryotic organisms__a
simple unicellular organism with no nuclear membrane, mitochondria, Golgi bodies,
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or endoplasmic reticlum that reproduces by asexual division. Although the cell wall
encircling bacteria is itself complex, there are two basic forms: a gram-positive cell
wall with a thick peptidoglycan layer and a gram-negative cell wall with a thin
peptidoglycan layer and an overlaying outer membrane. Some bacteria lack this cell
wall structure and compensate by surviving only inside host cells or in a hypertonic
inviroment. The human body is initiated by thousands of different bacterial
species__some living transiently, others in a permanent parasitic relationship.
Likewise, the enviroment that surrounds us, including the air we breathe, water we
dring, and food we eat, is inhabitated by bacteria, many of which are relatively
avirulent and some of which are capable of producing life-threatening disease.
Fungi
In contrast to bacteria, the cellular structure of fungi is more complex. These are
eukaryotic organisms that contain a well-defined nucleus, mitochondria, Golgy
bodies, and endoplasmic reticulum. Fungi can exist either in unicellular form (yeasts)
that can replicate asexually or in multycellular (molds) that can replicate asexually
and sexually.
Parasites
Parasites are the most complex microbes. Although all parasites are classified as
eukaryotic, some are unicellular and others are multicellular. They range in size from
tiny protozoa as small as 1 to 2 µm in diameter ( the size of many bacteria) to
arthropods and tapeworms that can measure up to 10 m in length. Indeed, considering
the size of some these parasites, it is hard to imagine how these organisms came to be
classified as microbes. Their life cycles are equally complex, with some parasites
establishing a permanet relationship with humans and others going through a series of
developmental stages in a progression of animal hosts.
MEDICAL BACTERIOLOGY
(fungi and algae) or may not (protozoa trophozoites) contain cell wall. On the other
hand, the eucaryotic cells can be either unicellular or multicellular.
Procaryotic cells are smaller and generally less complex than eucaryotic cells.
They are unicellular, have neither a well-defined nucleus nor nuclear membrane,
undergo amitotic division, and (with one exception) contain a cell wall of unique
chemical composition. Other components like; endoplasmic reticulum, mitochondria,
and lysosomes are not found.
COCCUS
A: Diplococcus B: Streptococcus C: Staphylococcus J: Tetrate
BACILLUS
D: Bacillus anthracis E: Haemophilus influenzae F: Fusiform G : Flamentous
H: Vibrio(curved) I: Spiral
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A. Cell Envelope
Cytoplasmic Membrane
The cytoplasmic membrane is a thin, ductile, and elastic membrane that
encloses the cytoplasm and is composed mostly of proteins and lipoproteins
embedded in a phospholipid bilayer. This membrane functions as a significant
osmotic barrier to low molecular weight substances, and as a semipermeable
membrane controlling the exchange of substances between the cell and surrounding
medium. Several enzimatically mediated biosynthetic processes, as well as electron
transport and oxidative phosphorylation, take place within the membrane.
Mesosomes are intracellular membrane structures, formed by an invagination of
the cytoplasmic membrane. They are more frequently seen in Gram-positive than
Gram-negative bacteria. Mesosomes present at the septum of Gram-positive bacteria
are involved in chromosomal separation; at other site they may be associated with
cellular metabolism.
Cell Wall
The cell wall is a constituent of all bacteria, except members of the genus
Mycoplasma. This structure confers rigidity and shape to the bacterial cell and acts as
a barrier to low molecular weight substances. Inasmuch as mammalian cells lack cell
walls, antimicrobial agents that inhibit cell wall synthesis (e.g., penicillin) will have
sellective activity gainst bacteria and little or no host toxicity. Bacterial rigidity and
shape are attriutable to layer, which lies closest to the cytoplasmic membrane and is
referred to as peptidoglycan (syn: murein, mucopeptide).
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According to the structural and biochemical aspects of their cell wall, there are two
groups of bacteria (Fig. 3):
1. Gram Pozitive Bacteria: The walls of gram-pozitive (G+ ) organisms are thick,
compact, and almost exclusively peptidoglycan. All gram-pozitive bacteria contain
lipoteichoic acid (LTA) convalently linked to the cytoplasmic membrane, and as
major surface antigens, may acts as a virulence factors. Several gram-positive
bacteria have surface proteins that are linked to either the cytoplasmic membrane or
peptidoglycan, and may acts as virulence factors or serve as the basis for a
clasification scheme.
2. Gram Negative Bacteria: The cell wall of gram-negative bacteria are thinner, less
compact, and more complex in their chemical composition. They are composed of a
distinct outer membrane bilayer and a distinct, thin peptidoglycan layer separated
from the cytoplasmic membrane by a periplasmic space. The outer membrane is
composed essentially of lipoproteins, phospholipids, porins, and lipopolysaccharides.
Lipoproteins are the most abundant proteins of gram negative bacteria; the lipid
end is insrted into the outer membrane while the protein end is linked to
peptidoglycan.
Phospholipids from outer membrane matrix and contribute to outer membrane
stabilization.
Porins are outer membrane trimers that form channels that permit small molecular
weight solutes to diffuse across the outer membrane; because porins are penetrated
slowly by large molecules, they may contribute to the relatively high resistence of
gram negative organisms to certain antimicrobial agents.
Lipopolysaccharide (LPS) (syn: endotoxin) consist of glycolipid complex,
referred to as lipid A, linked to a polysaccharide. LPS is toxic for humans, producing
hypotension, fever, shock, intravascular coagulation, and/or tissue necrosis. The lipid
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A is responsible for the toxicity of the molecule, the core polysaccharide components
constitute antigenic region ( O antigen ) to many gram negative organisms, and
consist of major surface antigens with diverse epitopes that confer specificity upon
the organisms.
Protoplast refers to a shapeless gram positive bacterium whose cell wall hase been
removed in a osmotically stabilized medium by lysosome hydrolysis or by inhibition
of cell wall synthesis with an antimicrobial agent such as penicillin. Similar treatment
of a gram negative bacterium results in a nearly cell wall-less organisms that has only
retained some or all of its outer membrane components and is referred to as a
spheroplast. Actively growing and multiplying protoplast and spheroplast are
generally called L forms.
Capsules
Capsules are well difined mucoid structures, usually polysaccaride in nature,
which closely surround the cell wall of some bacteria (Fig. 4). These capsules may be
antiphagocytic, vaccinogenic, and/or identifiable in rapid diagnostic tests utilizing
specific antiserum. Also, the dextran and levan capsules of Streptococcus mutans are
the means by which the bacteria attach and stick to the tooth enamel.
Bacterial Slime
Extracellular slime layers are produced by
some bacteria. They are more loosely bound to the cell surface than capsules and are
water soluble. The slime is composed of complex polysaccharides. It is a virulence
factor, e.g. facilitating the attachment of Staphylococcus epidermidis onto artificial
surfaces, such as intravascular cannulae and protect the organism from antibiotics and
host defences. The capsule and slime layers are also called the glycocalyx
Flagella
Flagella are composed entirely of a single protein subunit called flagellin,
which differs in primary structure among different bacterial speciese, and are
responsible for bacterial motility that may inhance bacterial invasion. The surface of
flagella is made up of protein antigens with diverse epitopes useful in the
identification and classification of organisms. They can be single (monotrichous) or
multiple (peritrichous). Spirochetes contain similar motility apparatus, protein in
nature, which leis in the periplasmic space between the cytoplasmic membrane and
the outer membrane.
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Pili
Pili are short, hairlike, rigid, surface appendages originating in the cytoplasmic
membrane. These appendages are found predominantly in gram negative organisms
and are composed of protein subunits called pillin. There are two types of pili,
referred to as sex (F) pili and common pili (fimbriae).
1. Sex pili. Sex pili, of which only 1-4 are found at random bacterial sites, mediate
the conjugation of donor and recipient cells and may participate in DNA transfer.
2. Common pili. In contrast, as many as 200 common pili may be evenly distributed
over the surface of an organism and may act as virulence factors by mediating
adherence to host cell surface, e.g. Neisseria gonorrhoeae produce fimbriae that
bind to specific receptors of cervical epithelial cells.
B. Intracellular Structure
The Cytoplasm
The cytoplasm is the complex mixture of substances enclosed by the envelope. It
consist of an amorphous equeous fluid in which are dissolved or suspended a myriad
of metabolites, enzymes, ions, ribosomes, plasmids, and storage granules. Embedded
in this fluid is a fibrous mass, called the nuclear material.
Nuclear Material
The bacterial material consists of a single circular molecule of double-stranded
DNA, about 1 mm long when unfolded. It is tightly packed within the bacterium and
is not surrounded by a nuclear membrane as in mammalian cells. Smaller extra-
chromosomal DNA molecules, called plasmids, that can replicate independently, may
also be present. Plasmids are most commonly found in gram-negative bacteria, and
although not usually essential for cellular survival, they often provide a selective
advantage: many confer resistance to one or more antibiotics.
Bacterial Ribosomes
The cytoplasm has many ribosomes which contain RNA and proteins, and are
involved in protein synthesis. These ribosomes are 70S monomers composed of 30S
and 50S subunits in contrast to the 80S monomer mammalian ribosomes that are
made up of 40S and 60S subunits.
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Cytoplasmic Granules
are located in the bacterial cytoplasm. These granules represent stored food
reserves consisting of protein, polysaccharide, and/or lipid. The size of theses
granules can increase in a favourable enviroment and decrease when conditions are
adverse, e.g. Corynebacterium diphtheriae may contain high energy phosphate
reserves in granules termed volutin granules.
Spores
BACTERIAL PHYSIOLOGY
Bacterial Growth
Most bacteria will grow on artificial culture media. However, some bacteria, e.g.
Mycobacterium leprae (leprosy) and Treponema pallidum (syphilis), cannot yet be
grown in vitro; other bacteria, e.g. chlamydiae and rickettsiae, only replicate within
host cells and are grown in tissue-culture.
Under suitable conditions (nutrients, temperature and atmosphere) a bacterial cell
will increase in size and then divide into two identical cells (binary fission). These
two cells are able to grow and divide at the same rates as the parent cell, providing
conditions remain stabe (Fig. 6). The time required for the number of bacteria in a
culture to double is called the generation time; e.g. Escherichia coli has a generation
time of about 30 min under optimal conditions.
All cells require a constant supply of energy to survive. This energy, typically in
the form of adenosine triphosphate (ATP), is drived from the controlled breakdown of
various organic substrates (carbohydrates, lipids, and proteins). This process of
substrate breakdown and conversion into usable energy is known as catabolism. The
energy produced may then be used in the synthesis of cellular constituents (cell walls,
proteins, fatty acids, and nucleic acids).
The metabolic process generally begins with hydrolysis of large macromolecules in
the external cellular enviroment by specific enzymes or exoenzymes. The small
subunit molecules produced (monosaccharides, short peptides, and fatty acids) are
transported across the cell membranes into the cytoplasm by active or passive
transport mechanisms specific for the metabolite. The metabolites are converted by
one or more pathways to one common universal intermediate, pyruvic acid. From
pyruvic acid the carbon may be channeled towards energy production or the synthesis
of a new carbohydrates, amino acids, lipids, and nucleic acids.
Bacteria are classified into two groups according to the type of compounds they
can utilize as a carbon source.
Autotrophs utilize organic carbon from carbon dioxide and nitrogen from
ammonia, nitrites and nitrates; they are of minor medical importance. Heterotrophs
require organic compounds as their major source of carbon and energy; they include
most bacteria of medical importance. Heterotrophs may have very complex or very
simple requirements for organic molecules. Bacteria must also have access to the
major elements sulfur, phosphorus, potassium, calcium, magnesium, and iron, plus
some minor elements. Most bacteria require varying numbers of growth factors,
which are organic compounds required by the cell in order to grow, but which the
organism cannot itself synthsize (for example, vitamins).
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2. Atmospheric Conditions
Carbon dioxide (CO2). Bacteria require CO2 for growth; adequate amounts are
present in air or are produced during metabolism by the organisms themselves. A few
bacteria require additional CO2 for growth, e.g. Neisseria meningitidis.
Oxygen. Bacteria may be classified into four groups according to their oxygen
requirements:
-obligate (strict) aerobe grow only in the presence of oxygen, e.g. Pseudomonas
aeruginosa;
-microaerophilic bacteria grow better in low oxygen concentrations, e.g.
Campylobacter jejuni;
-obligate (strict) anaerobe grow only in the absence of free oxygen, e.g.
Bacteroides spp.;
-facultative anaerobes grow in the presence or absence of oxygen, e.g. E.coli.
pH. Most pathogenic bacteria grow best in slightly alkaline pH (pH 7.2-7.6). A few
exceptions exist; Lactobacillus acidophilus, present in the vagina of postpubescent
females, prefers an acid medium (pH 4.0). It produces lactic acid which keeps the
vaginal secretions acid, thus preventing many pathogenic bacteria from establishing
infection. Vibrio cholerae, the cause of cholera, prefers an alkaline enviroment (pH
8.5).
3. Energy production
When bacteria are added (inoculated) into a liquid growth medium, subsequent
multiplication can be followed by determining the total number of viable organisms
(viable counts) at various time intervals. The growth curve produced normally has
four phases (Fig. 7):
Lag phase of growth occurs while the cell adapts to its new surroundings. During
this time, the metabolic activity of the organism is increased in preparation for
bacterial division. An increase in bacterial RNA and protein synthesis is noted, as is
an increase in the size of the bacterial particles.
Exponential or log phase of growth, the next step in bacterial proliferation,
represents the rapid cell growth and division. During this stage bacteria are especially
susceptable to agents such as antibiotics, probably because the physiologic effeciency
provides more frequent opportunities for interaction with the antibiotic and the
bacterial envelope barriers are more easily traversed.
Stationary growth phase of growth occurs when some essential nutrient becomes
limiting in the bacterial inviroment, or alternatively, a toxic end product of cell
metabolism accumulates that is deleterious for the growth process. Cell growth then
slows and usually stops within a single cell generation time.
If new or alternative nutrients are not encountered, cellular energy supplies are
eventually depleted and the cells enter a death phase.
Liquid media can be solidified with agar which is extracted from algae. A
temperature of 100 ºC is used to melt agar, which then remains liquid until the
temperature falls to approximately 45 ºC, when it produces a transparent gel. Solid
media are normally set in Petri dishes (agar plates). Most bacteria grow on solid
media to produce colonies. Each colony comprises thousands of bacterial cells which
emanated from a single cell. The morphology of the colony assists in bacterial
identification.
To culture bacteria in vitro, the microbiologist has to take into account the
physiological requirements. Various types of liquid and solid madia have been
developed for the diagnostic laboratory.
Simple media. Many bacteria will grow in simple media, e.g. nutrient
broth/nutrient agar which contains “peptone” (polypeptides and amino acids from the
enzymatic digestion of meat) and “meat extract” (water-soluble components of meat
containing mineral salts and vitamins).
Enriched media. These contain additional nutrients for the isolation of fastidious
bacteria which require special conditions, e.g. blood agar; chocolate agar (heated to
lyse erythrocytes and release additional nutrients).
Selective media. These are designed to facilitate growth of some bacteria, whilst
suppressing the growth of others and include: MacConkey agar, which contains bile
salts and allows the growth of bile-tolerant bacteria only; alkaline peptone broth,
which allows selective growth of vibrios which prefer alkaline conditions; and
antibiotics, which are frequently added to media to allow only certain bacteria to
grow whilst suppressing or killing others.
Indicator media. These are designed to aid the detection and recognition of
particular pathogens. They are often based on sugar fermentation reactions which
result in production of acid and the subsequent colour change of pH indicator, e.g.
MacConkey agar contains lactose and a pH indicator (neutral red); lactose-fermenting
bacteria (e.g. E.coli) produce acid and form pink colonies, whilst non-lactose
fermenting bacteria (e.g. salmonella) do not produce acid and form pale yellow
colonies. This property facilitates the recognition of possible salmonella colonies
amongst normal bowel flora.
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BACTERIAL GENETICS
The bacterial genome is the total collection of genes (2000 to 4000 in number)
carried by a bacterium both on it is chromosome and on it is extrachromosomal
genetic elements, if any. The bacterial chromosome differs in several ways from the
human chromosome:
Plasmids are small genetic elements that replicate independently of the bacterial
chromosome. Most plasmids are circular, double stranded DNA molecules varying
from 1500 to 400,000 base pairs. Like the bacterial chromosomal DNA, they can
autonomously replicate and as such are referred to as replicons. Some plasmids, such
as the E.coli F plasmid, are episomes, which means that they can integrate into the
host chromosome.
Plasmids carry genetic information, which may not be essential but can provide a
selective advantage to the bacteria. For example: Antibiotic resistance, production of
toxins and metabolizing some substrates.
Bacteriophages (phages) are viruses that replicate inside of the bacterial cells.
These extrachromosomal genetic elements can survive outside of a host cell because
the nucleic acid genome (which may be DNA or RNA) is protected by a protein coat
(Fig. 8). The typical replicative cycle begins with attachment of the phage to
receptors on the cell surface, followed by injection of the nucleic acid into the
bacterial cell, leaving all or most of the protein outside the cell (this is incontrast to
viral infection of vertebrates cells, where the entire virus is taken up by the cell, and it
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is nucleic acid released intracellularly). The phage nucleic acid takes over the cell’s
biosynthetic machinery to replicate it is own genetic material, and to synthesize
phage-specific enzymes and proteins of the phage coat. When sufficient coat proteins
and new phage have accumulated, these components self-asemble into mature phage
particles with the DNA encapsulated by the phage coat. Release of the new phage
particles is accomplished by a phage-specific enzyme (a lysozyme) that disolves the
bacterial cell wall.
Fig. 8 Bacteriophage
Protein Synthesis
The synthesis of protein is primarily controlled by DNA. The design of each proteins
is trasmitted from DNA to mRNA, which instructs the cellular machinery to assemble
a particular protein. Proteins serve structural and enzymatic purposes and thereby
determine the structure and metabolic function of any organism. The total genetic
potential of DNA constitute the genotype of the bacterium; what actually becomes
manifest discernible to the observer is the phenotype.
The bacterial genome consist of 3,000 to 6,000 genes-distinct DNA sequences that
specifiy the sequence of amino acids in polypeptide chain. Each gene determines a
particular kind of amino acid assembly. The chain of nucleotides that constitute a
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gene is composed of groups of bases. Each set of three bases, known as a codon,
specifies a particular amino acid. Since reading of the massage can begin at any base,
three different transcriptions would be possible.
GENETIC VARIATION
Mutation
During replication of DNA, copying errors called mutations may occur leading to
changes in the sequence of nucleic acids. Many mutations occur spontaneously in
nature (e.g., polymerase mistakes); however, mutations can also be induced by
physical or chemical agents. Among the physical agents used to induce mutations in
bacteria are heat, ultraviolet, and x-rays. Chemicals that are mutagens are nucleotide-
base analogues, acridine derivatives, and DNA-reactive chemicals.
Mutations can be classified according to the kind of chemical change that occurs in
the DNA:
1. Base substitutions: A single base change can result in a transition, in which
one purine (A or G) is replaced by another purine or in which a pyrimidine (C
or T) is replaced by another pyrimidine. A transversion, in which for example,
a purine is replaced by a pyrimidine and vice versa, may also result.
2. Additions and deletions: A single base pair, or a contiguous string of base
pairs, can be added to or deleted from the DNA.
More major changes which lead to significant alterations in the organism, are often
detrimental and the mutant organism may not survive. However, under certain
circumstances, such alteration can result in a mutant cell with a significant advantage,
allowing it to autgrow other daughter cells; for example, antibiotic resistance mutants
may be selected out when that particular antibiotic is present in the enviroment.
Transformation
Transformation is the process by which bacteria take up fragments of naked DNA
from the surroundings and incorporate them into their genome. This process of
transformation was first described in Streptococcus pneumoniae. How DNA gets
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across the bacterial membrane is not well understood. It is clear, however, that some
bacteria have special receptors for DNA on their membranes, which facilitate the
internalization of DNA.
Transformation appears to be major source of genetic exchange for a number of
bacteria including N.gonorrhoeae and S.pneumoniae. It is widely exploited by
scientists and genetic engineers to introduce recombinant DNA forms.
Conjugation
Conjugation refers to the process by which bacteria transfer genes from one cell to
another by means of cell-to-cell contact. Specifically, the process requires the
presence of the donor cell of hair-like projrctions called sex pili that make contact
with specific receptor sites on the surface of the recipient cell.
A plasmid isolated from E.coli called the F (fertility) factor has been intensively
studiet and serves to illustrate the process of conjugation. It is related to plasmids
moved from cell to cell by conjugation. Only bacteria that carry the F vactor (denoted
as F+) produce sex pili, which can attach specifically to a bacterium that does not
carry the F factor (denoted as F-). In a process that is not well understood, the pili
bring the cells close together, a bridge is formed between the two cells, and the F
factor DNA is copied and transferred from the F+ to the F- cell, converting it to an F+
cell (Fig. 10).
The transfer of DNA is also directed by a set of genes carried on the F plasmid.
First, the plasmid is cleaved on the strand at a specific site. Next, an end of DNA
produced bu this cleavage enters the recipient cell and continues to move across until
the entire strand is transferred, at which the points ends are joinned to form a circular
molecule. This single-strand molecules is then converted to double-stranded DNA by
the action of DNA polymerase. During this process, the complementary strand of
DNA remains within the donor cell and is also converted to duplex DNA. In this way,
the plasmid is transferred to a new cell while the donor cell maintains its copy.
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Occasionally, the F factor will integrate itself into the bacterial chromosome by
homologous recombination (Fig. 11). When this happens, part and sometimes all of
the bacterial chromosome can be transferred to a recipient cell by way of the pathway
discussed above. Such cells are denoted Hfr (high frequency of recombination) to
indicate that they often transfer chromosomal genes to recipient cells. Conjugation is
a particularly devise for transferring plasmids (and for Hfr strains, chromosomal
genes as well) from one bacterium to another (Fig. 12).
Plasmids are used extensively in recombinant DNA technologies. Foreign DNA
(e.g., from humans) can be inserted into bacterial plasmids where it can be easily
reproduced and manipulated.
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Transduction
Transduction refers to transfer of genes from one cell to another via a phage vector
without cell-to-cell contact. There are two ways in which this occur:
Specialized transduction
Upon infection, temprate viruses can reproduce and lyse the host cell as described
above. These viruses also have an alternative pathway available to them: They can
insert DNA into the host chromosome, causing the bacterium no harm. Such a virus
might remain dormant in the bacterium for many generations. However, in response
to certain enviromental signals, the dormant virus can induced to lytically, killing the
host cell and releasing hundreds of progeny phage. The temprate phage, in gaining its
independence from the bacterial chromosome, may carry with it small pieces of
donor bacterial DNA, which, when delivered to the next host cell, add a new attribute
to the new host’s capabilities.
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GENETIC ENGINEERING
Pathogen: An organism which can invade the body and cause disease
Infection: A disease caused by pathogen.
Pathogenicity: The ability to cause disease.
Virulence: The pathogen’s power to cause severe disease.
Epidemiology: The study of the distribution and determinants of diseases in
populations.
Opportunistic microorganism: Is an organism that is unable to cause disease in
healthy, immunocompetent individuals, but can infect people whose specific or
nonspecific defenses have been impaired.
Microbial Flora
Medical microbiology is the study of the interactions between humans and
microorganisms such as bacteria, viruses, fungi, and parasites. Although are primary
interest is in diseases caused by these interactions, it must also be appreciated that
microorganisms play a critical role in human survival. The normal commensal
population of microbes participates in the metabolism of food products, provides
essential growth factors, protects against infections with highly virulent
microorganisms, and stimulates the immune response. In the absence of these
organisms, life as we know it would be impossible.
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The microbial flora in and on the human body is in a continual state of flux
determined by a variety of factors, such as age, diet, hormonal state, health, and
personal hygiene. Whereas the human fetus lives in a protected, sterile enviroment,
the newborn is exposed to microbes from the mother and enviroment. The infant’s
skin is colonized first, followed by the oropharynx, gastrointestinal tract, and other
mucosal surfaces. Throughout the life of an individual, this microbial population
continues to change. Changes in health can drastically disrupt the delicate balance
that is maintained among the heterogeneous organisms coexisting within as. For
example, hospitalization can lead to the replacement of normally avirulent organisms
in the oropharynx with gram-negative bacilli (e.g., Klebsiella, Pseudomonas) that can
invade the lungs and cause pneumonia.
Exposure of an individual to an organism can lead to one of three outcomes. The
organism can (1) transiently colonize the person, (2) permanently colonize the
person, or (3) produce disease.
An understanding of medical microbiology requires knowledge not only of the
different classes of microbes but also of their propensity for causing disease. A few
infections are caused by strict pathogens (organisms always associated with human
disease). Some examples of strict pathogens and the diseases they cause include
Mycobacterium tuberculosis, Neiseria gonorrhoeae, plasmodium spp., and rabies
virus. Most infections are caused by opportunistic pathogens (e.g., Staphylococcus
aureus, Escherichia coli, Candida albicans).
Ear
The most common organism colonizing the outer ear is coagulase-negative
Staphylococcus. Other organisms colonizing the skin have been isolated from this
site, as well as potential pathogens such as S.pneumonia, Pseudomonas aeruginosa,
and the Enterobacteriaceae.
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Eye
The surface of the eye is colonized with coagulase-negative staphylococci as well
as rare numbers of organisms found in the nasopharynx (e.g., Haemophilus spp,
Neisseria spp, and viridans streptococci).
Gastrointestinal Tract
Esophagus
Oropharyngeal bacteria and yeast, as well as the bacteria that colonize the stomach,
can be isolated from the esophagus.
Stomach
Because the stomach contains hydrochloric acid and pepsinogen, the only
organisms present are small numbers of acid-tolerant bacteria such as the lactic acid-
producing bacteria (Lactobacillus and Streptococcus spp.) and Helicobacter pylori (is
a cause of gastritis and ulcerative disease).
Small intestine
In contrast with the anterior portion of the digestive tract, the small intestine is
colonized with many different bacteria, fungi, and parasites. Most of these organisms
are anaerobes, such as Peptostreptococcus, Porphyromonas, and Prevotella. Common
causes of gastroenteritis (e.g., Salmonella and Campylobacter spp.) can be present in
small numbers as asymptomatic residents; however, their detection in the clinical
laboratory generally indicates disease.
Large intestine
More microbes are present in the large intestine than anywhere else in the human
body. It is estimated that more than 1011 bacteria per gram of feces can be found, with
anaerobic bacteria in excess by more than 1000-fold. Various yeasts and
nonpathogenic parasites can also establish residence in the large intestine. The most
common bacteria include Bifidobacterium, Eubacterium, Bacteroides, Enterococcus,
and the Enterobacteriaceae. E.coli is present in virtually all humans from birth until
death. Although this organism represents less than 1% of the intestinal population, it
is the most common aerobic organism responsible for intra-abdominal infections.
Antibiotic treatment can rapidly alter the population, causing the proliferation of
antibiotic-resistant organisms such as enterococci, Pseudomonas, and fungi.
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Genitourinary System
In general, the anterior urethra and vagina are the only anatomic areas of the
genitourinary system permanently colonized with microbes. Although the urinary
bladder can be transiently colonized with bacteria migrating upstream from the
urethra, these should be cleared rapidly by the bactericidal activity of the
uroepithelial cells and the flushing action of voided urine.
Anterior urethra
The commensal population of the urethra consists of a variety of organisms,
lactobacilli, and coagulase-negative staphylococci the most numerous. These
organisms are relatively avirulent and are rarely associated with human disease.
Vagina
The microbial population of the vagina is more diverse and is dramatically
influenced by hormonal factors. Newborn girls are colonized with lactobacilli at
birth, and these bacteria predominate for aproximately 6 weeks. After that time, the
levels of maternal estrogen have declined, and the vaginal flora changes to include
staphylococci, streptococci, and Enterobacteriaseae. When estrogen production is
initiated at puberty, the microbial flora again changes. Lactobacilli reemerge as the
predominant organisms, and may other organisms are also isolated, including
staphylococci, group B streptococci, Enterococcus, Gardnerella, Mycoplasma,
Ureaplasma, and a variety of anaerobic bacteria.
The lactobacilli metabolize the glycogen of the vaginal epithelium to produce
lactic acid, resulting in a low pH that limits some potential pathogens.
Skin
Although many organisms come to contact with the skin surface, this relatively
hostile enviroment does not support the survival of most organisms. Gram-positive
bacteria (e.g., coagulase-negative Staphylococcus, corynebacteria, and
propionibacteria) are the most common organisms found on the skin surface. Fungi
Candida is also found on skin surfaces, particularly in moist sites. Gram-negative
bacteria do not permanently colonize the skin surface because the skin is too dry.
STERILIZATION
Moist and Dry heat are the most common sterilizing methods used in hospitals and
are indicated for most materials except those that are heat-sensitive or consist of toxic
or volatile chemicals.
Dry heat
Dry heat destroys microorganisms by oxidation. There are three types of dry heat
sterilization: flaming to red heat, incineration and hot-air ovens.
Incineration is considered the best method to dispose of infected carcasses and
organic wastes; sterilization of inoclating loops by passage through a bunsen burner is
another effective form of incineration.
Flaming to red heat. Metal instruments, such as dental reflection mirrors, can be
sterilized by direct heating. In an emergency, scalpels can be sterilized by dipping the
blade in methylated spirit and burning off the spirit.
Hot-air ovens produce dry heat by gas or electricity in insulated double walled
metal containers. The temperature and time required for effective sterilization is
171ºC for 1 hour, 160ºC for 2 hour or 121ºC for 16 hour. The process is useful on
heat stable materials and on substances that steam cannot penetrate such as oil.
The effectiveness is monitored with spore tests using Bacillus subtilis, which is
relatively resistant to killing by dry air (in contrast with Bacillus stearothermophilus).
28
Moist Heat
Sterilization by moist heat is more rapid and efficient than dry heat; the presence of
water causes protein denaturation resulting in disruption of cell membranes, and
improves heat penetration.
There are three types of moist heat sterilization: the autoclave, pasteurization, and
boiling water.
Boiling water, even for as long as 30 min, is not recommended for sterilization,
because it does not kill bacterial spors or certain viruses such as hepatitis B.
Ultraviolet Light
Sterilization by X- and gamma rays is used for delicate supplies such as syringes
and sutures, as well as increasingly for food. Radiation kills bacteria and other life
forms by damaging most cell constituents, including DNA, usually by free radical
formation.
Filtration
DISINFECTION
ANTISEPSIS
Antiseptic agents are used to reduce the number of microbes on skin surfaces.
These compounds are selected for their safety and efficacy.
Alcohols (70 to 90%) have excellent activity against all groups of organisms
except spores and are nontoxic, although they tend to dry the skin surfaces because
they remove lipids. They also do not have residual activity and are inactivated by
organic matter. Thus, the surface of the skin should be cleaned before alcohol is
applied.
Iodophors ( 1 to 2% available iodine) are also excellent skin antiseptic agents,
having a range of activity similar to that of alcohols. They are slightly more toxic to
the skin than is alcohol, have limited residual activity, and are inactivated by organic
matter. Iodophors and iodine preparations are frequently used with alcohols for
disinfecting the skin surface.
Chlorhexidine (0.5 to 4%) has broad antimicrobial activity, although it kills
organisms at a much slower rate than does alcohol. Its activity persists, although
organic material and high pH levels decrease its effectiveness.
The activity of parachlorometaxylenol is limited primarily to gram-positive
bacteria. Because it is nontoxic and has residual activity, it has been used in
handwashing products.
Triclosan is active against bacteria but not against other organisms. It is a common
antiseptic agent in deodorant soaps and some toothpaster.
Chemicals
Avariety of chemicals are used for disinfection. They are evaluated as to whether
they kill vegetative bacteria, mycobacteria, Pseudomonas, fungi, or spores.
Surface-Active Agents
These agents injure the bacterial cell by damaging the cytoplasmic membrane and
altering cell permeability
b. Anionic detergents are soap and fatty acids that dissociated to yield negatively
charged ions that disrupt cytoplasmic membrane lipoproteins.Their inability to kill
spores or several gram-negative organisms associated with nosocomial (hospital
acquired) diseases renders them of little value as disinfection.
Phenoles
Are compounds that bind to and denature proteins within the bacterial cytoplasmic
membrane resulting in membrane damage, leakage of cell contents, and lysis the of
the organism. These compounds are bactericidal for both gram negative and gram
positive bacteria, including the spores formers. Phenolic compounds, such as
AmphylR in concentrations of 2-5%, are excellent disinfectants for washing down
surfaces such as operating room floor and laboratory benches.
Alcohols
Denature cell proteins and disorganize cytoplasmic membrane lipids resulting in a
loss of membrane permeability. Alcohols are rapidly bactericidal against vegetative
bacteria, mycobacteria, some fungi, and lipid-containing viruses. Unfortunately,
alcohols have no activity against bacterial spores and have poor activity against some
fungi and non-lipid-containing viruses. Activity is greater in the presence of water.
Thus, 70% alcohol is more active than is 95% alcohol.
Halogens
Halogens such as compounds containing iodine or chlorine, are used extensively as
disinfectants.
Iodine compounds are the most effective halogens available for disinfection.
Iodine is a highly reactive element that precipitate proteins and oxidizes enzymes. It
is microbicidal against vertually all organisms, including spore-forming bacteria and
mycobacteria. Elemental iodine can be dissolved in aqueous potassium iodide or
alcohol, or it can be complexed with a carrier. The latter is reffered to as an iodophor.
Povidone iodine is used most commonly, is relatively stable and nontoxic to tissues
and metal sufaces, but is expensive compared with other iodine solutions.
Chlorine compounds are are also used extensively as disinfectants. Three forms
of chlorine may be present in water: elemental chlorine (Cl2), which is very oxidizing
agent; hypochlorous acid (HOCl); and hypochlorite ion (OCl2). Chlorine also
combines with ammonia and other nitrogenous compounds to form chloramines or N-
chloro compounds. These compounds demonstrate good germicidal activity, although
spore-forming organisms are 10- to 1000-fold more resistant to chlorine than are
vegetative bacteria.
Oxidizing Agents
Examoles of oxidants include ozone, peracetic acid, and hydrogen peroxide, with
the last used most commonly. Hydrogen peroxide effectively kills most bacteria at a
concentration of 3 to 6% and kills all organisms including spores, at higher
32
Heavy Metals
Inactivate microbial enzyme systems resulting in interference with protein
synthesis. Most heavy metals are too toxic for human use, but some have been
employed prophylactically with seccess.
Ethylene oxide
Ethylene oxide is a colorless gas, soluble in water and comming organic solvents,
that is used to sterilize heat sensitive items. It interferes with protein synthesis by
alkylating protein and thus blocking free amino groups. It is an excellent bactericidal
agent against gram-negative and garm-positive organisms, including spore formers,
and is used in the disinfection of hear-lung machines, polyethylene tubing, lensed
instruments, biologicals, and other materials that would otherwise be damaged by
heat or disinfecting solutions. Sterilization by ethylene oxide can be acchomplished
by using 450 to 700 mg of ethylene oxide per liter of chamber space at 55 to 60ºC for
two hours. Care must be taken when using ethylene oxide due to its potential tissue
toxicity. Although a 24 to 72 h. period of aeration is required after disinfection in
order to get rid of residual fumes.
Formaldehyde
is a gas whose mechanism of action and bactericidal activity is identical to that of
ethylene oxide. It is used to decontaminate rooms and fabrics, but causes damage to
the cutting edges of instruments. Like ethylene oxide, it is irritating to the skin and
leaves residual fumes which necessitate similar periods of aeration. Formalin is
formaldehyde in aqueous form. It is used at a 10% concentration to preserve tissues
for histopathological study. At appropriate concentration, formalin is valuble in the
perparation of several vaccines by virtue of it is ability to convert toxins to nontoxic,
and vaccinogenic toxoids.
Glutaraldehyde
It is an alkylating agent that binds sulfhydryl or amino groups. In 2% aqueous
solution, it is an excellent bactericidal agent against gram-negative and gram-positive
organisms, including spore formers. Glutaraldehyde is highly effective disinfectant
for lensed and other surgical instruments.
33
ANTIMICROBIAL AGENTS
Until the twentieth century, there were no special agents available to treat
infectious diseases, other than folk remedies and herbal concoctions. Amazingly, the
hundreds of antimicrobial agents available today were developed within the last 80
years. In 1924, Fleming first isolated penicillin from penicillium notatum. It was
introduced to clinical medicine during World War II by Chain and Florey and
abruptly changed the practice of medicine. Later, in the 1940s and 1950s,
streptomycin and the tetracyclines were developed and were followed rapidly by
additional aminoglycosides, cephalosporins, quinolones, and other antimicrobials.
Criteria for a successful antimicrobial agent are listed below:
1. Displays targeted toxicity to the offending microbe only.
2. Produces no side effects in the host.
3. Displays a narrow range of activity and does not harm normal host flora.
4. Kills the targeted microorganism (bactericidal), rather than merely inhibiting its
growth (bacteriostatic).
5. Fails to induce resistence.
6. Is soluble in body fluids and tissues.
Obviously, this ideal has not yet been met. In most cases, the goal of
antimicrobial therapy is to reduce the total numbers of infecting microbes to a
level low enough that host defenses can destroy them.
Definitions
Antimicrobial Agent: A substances that either kills, inhibits growth of, or prevents
damage due to an infectious microorganism. Antibacterial, antifungal, antiprotozoal,
antihelminthic, and antiviral agents are subsets of antimicrobial agents.
Antibiotic: A substance produced by a fungus or bacterium that kills or inhibits
growth of other microorganisms.
Chemotherapeutic Agent: A substance, synthetically derived, used to treat any
disease, infectious or non-infectious (e.g., cancer or hypertension).
.....cidal: Indicate that the action of the agent will kill the targeted microbe.
Bactericidal, fungicidal, and virucidal are derived terms.
.....static: Indicates that the action of the agent will inhibit the growth of the
targeted microbe but will not kill it. Bacteriostatic, and fungistatic are derived terms.
Pharmacokinetics: The study of the concentration and activities of
chemotherapeutic agents in patients.
Synergy: The antimicrobial activity of two antimicrobial agents used together is
greater than the additive effect of both agents used alone.
Antagonism: The antimicrobial activity of one antimicrobial agent is diminished
by another agent being administered concurrently.
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Penicillins
Penicillin compounds are highly effective antibiotics with extremely low toxicity.
The base compound is an organic acid with a ß-lactam ring obtained from culture of
the mold Penicillium chrysogenum.
Penicillin derivatives are achieved by adding or modifying side chains to the
structure of the basic ß-lactam ring:
1. Penicillin G.
Penicillin G is a narrow spectrum activity includes gram-positive cocci such as
streptococci (including S.pneumoniae, S. pyogenes, S. agalactiae), non-penicillinase
producing staphylococci, and certain gram-negative cocci such as Neisseriae spp..
Also it is effective against spirochetes and some anaerobic bacteria. On the other
hands, penicillin G, is ineffective against bacterial spp. that produced enzyme of
penicillinase (or beta lactamase). Penicillin G is incompletely absorbed because it is
inactivated by gastric acid. Thus it is used mainly as parenteral drug for serious
infections with penicillin-sensitive organisms.
The toxicity of penicillin G is extremely low, hypersensitivity to penicillin is
commonly present. 1% to 8% of the general population are allergic to the penicillins.
The hypersensitivity reactions range from immediate anaphylactic reactions to late
manifestations such as a skin rash.
Cephalosporins.
The cephalosporins are beta-lactam antibiotics derived from 7-
aminocephalosporanic acid, which was originally isolated for a Cephalosporium
mold. The antibiotics have the same mechanism of action as the penicillin but have a
wider antibacterial spectrum, are resistant to many beta-lactamase, and have
improved pharmacokinetic properties. The antimicrobial agents are divided into
“generations” based on their structure and spectrum of activity:
Polymyxins. The polymyxins are basic peptides (derived from Bacillus polymyxa)
that act as cationic detergents to cause lysis of the lipoprotein cell membrane.
Although polymyxin B and colistin are active against gram-negative bacteria
(including Pseudomonas), serious nephrotoxicity has limited their internal use. They
are used chiefly to treat local infections such as external otitis, eye infections, and
skin infections with sensitive organisms.
Tetracyclines.
The tetracyclines are broad-spectrum, bacteriostatic antibiotics that inhibit protein
synthesis in bacteria by blocking the binding of tRNA to the 30S ribosomal subunit.
Tetracyclines (e.g., tetracycline, doxycycline, minocycline) are effective in the
treatment of infections caused by Chlamydia, Mycobacteria, Rickettsia, and other
selected aerobic and anaerobic gram-positive and gram-negative bacteria. They are
absorbed rapidly from the gastrointestinal tract, and distribute widely in most fluids
and tissues, localizing particularly in bones and teeth. Because the tetracyclines will
37
Chloramphenicol.
Chloramphenicol, a broad-spectrum, bacteriostatic antibiotic, inhibits bacterial
protein synthesis by acting primarily on the 50S ribosomal unit. It is active against
large number of gram-positive and gram-negative organisms, rickettsiae and some
chlamydia, but is considered the drug of choice only for treatment of typhoid fever.
The reason for this is, in addition to interfering with bacterial protein synthesis,
chloramphenicol disrupts protein synthesis in human bone marrow cells and can
produce aplastic anemia.
Macrolides
The macrolides are a group of related antimicrobials. They have a common
macrocyclic lactam ring. The members of this group include erythromycin,
clarithromycin, azihromycin and spiramycin. All the agents have similar
antimicrobial spectrum, including gram-positive organisms, Neisseria, Haemophilus
and Bordetella, and some gram-negative anaerobes. They are also active against
Mycoplasma sp., Rickettsia sp. And Toxoplasma gondii. These agents disrupts protein
synthesis by binding to the 50S ribosomal subunit. Erythromycin, is used mainly to
treat pulmonary infections caused by Mycoplasma, Legionella, and gram-positive
organisms in patients allergic to penicillins.
Clindamycin
Also blocks protein synthesis by binding to the 50S ribosomal subunit. It is active
against staphylococci and anaerobic gram-negative bacilli but generally inactive
against aerobic gram-negative bacteria. Clindamycin can be administered orally or
intravenously with good penetration into tissues such as bone. Although intravenous
administration of clindamycin is associated with relatively few side effects, oral
administration can be responsible for gastrointestinal disturbances ranging from mild
diarrhea to life-threatining pseudomembranous colitis.
Rifampin
Asemisynthetic derivative of rifamycin B produced by Streptomyces mediterranei,
bactericidal for Mycobacterium tuberculosis and is very active against aerobic gram-
positive cocci. Because resistance can develope rapidly, rifampin is usually combined
with one or more other effective antibiotics. The drug inhibits DNA-dependent RNA
polymerase.
38
Quinolones (Fluoroquinolones)
A group of orally effective antibacterial agents, are chemically related to nalidixic
acid. The number of agens in this group of drugs has risen exponentially such as,
ciprpfloxacin, Enoxacin, ofloxacin, lomefloxacin and others. They are bactericidal
against most gram-negative organisms and many gram-positive organisms.
The mechanism of action of the quinılınes is unique and involves antagonism of
the DNA gyrase; the enzyme is involved in DNA synthesis.
Sulfonamides
Strictly speaking, the sulfonamides cannot be classified as antibiotics because they
are not produced by living organisms. They may be termed antiinfective or
antimicrobials.
The introduction of many newer antibiotics limited the use of sulfanamides until
the introduction of trimethoprim-sulfomethoxazole combination. This combination,
the most common use of the sulfonamides.
Sulfonamides inhibit the synthesis of folic acid in bacteria. Folic acid is required
for DNA synthesis. The sulfanamides are similar in structure to para-aminobenzoic
acid (PABA) and compete with PABA, then they inhibit the synthesis of folic acid
that the most important in the synthesis of bacterial DNA.
The sulfonamides are bacteriostatic against many gram-positive and som gram
negative bacteria. The combination of sulfomethoxazole with trimethoprim (SMX-
TMP) shows synergistic activity against a widw variety of gram-positive bacteria and
some gram-negative bacteria such as Enterobacteriaceae, Pseudomonas spp.,
Haemophilus influenzae, S.aureus, S.pyogenes, and S.pneumoniae.
SMX-TMP is indicated in the treatment of selected urinary tract infections and
selected respiratory and gastrointestinal infections.
BACTERIAL RESISTANCE
1.synthysizing en enzyme that destroys the antibiotic (e.g., the beta-lactamase that
cleaves the beta-lactam rings of penicillin and cephalosporin.
HOST-PARASITE RELATIONSHIPS
ECOLOGY
NORMAL FLORA
The human body is colonized on the skin and mucosal surfaces with a large
number of microorganisms which form the body’s normal flora. These organisms, far
from causing disease, often provide benefit to the host, by competing with potential
pathogens for attachment sites, by producing antimicrobial substances toxic to
pathogens and by competing for nutrients with pathogens. Reduction in normal flora
(e.g. by antibiotic therapy) may result in overgrowth of potential pathogens.
PATHOGENIC MICROORGANISMS
Pathogen: Defined as any organism capable of invading the body and causing
disease.
Opportunist: A microorgganism is not capabale of causing disease in an
immunologically and physically intact host, but when the normal host defenses are
defected in some way, this organism can cause disease.
INFECTION
MICROBIAL STRATEDIES
Transmission
Ability to survive outside the host is an important factor in transmission of
organism. Upper respiratory tract viruses (e.g., rhinoviruses) survive poorly outsite
the host and successful transmission relies on the production of large quantities of
41
infectious particles. Other organisms can survive outsite the host, e.g. Mycobacterium
tuberculosis and do not require large numbers of infectious particles for efficient
transmission.
4. Wounds. An infected or colonized animal host can inject the etiologic agent. For
example, cat-scratch disease occur when a normal individual is scratched by the
family cat, whose claws are contaminated with the organisms (normal oral flora in
cats).
Attachment
Many organisms have specific factors that allow attachment to mucosal surfaces,
e.g. the cell surface lipoteichoic acid of group A streptococci and pili of
N.gonorrhoea.
42
Invassion
Invasive bacteria are those that enter host cells or penetrate mucosal surfaces,
thereby spreading from the initial site of infection. Invassion is facilitated by several
bacterial enzymes, the most notable of which are collagenase and hyaluronidase.
These enzymes degrade components of the extracellular matrix, thereby providing the
bacteria with easier access to host cell surfaces.
Bacterial Toxins
Some bacteria cause disease by producing toxins, of which there are two general
types: the exotoxins and the endotoxins.
a. Exotoxins: The exotoxins, which are proteins, are secreted by both gram-
positive and gram-negative bacteria. In many cases, the toxin gene is encoded on a
plasmid (tetanus neurotoxin of Clostridium tetani and enterotoxin of E.coli) or a
lysogenic phage (Corynebacterium diphtheriae and Clostridium botulinum). The
exotoxin proteins generally have two polypeptide components. One component is
responsible for binding the protein to the host cell, whereas the second component is
responsible for the toxic effect. In many cases, the toxin is completely responsible for
causing characteristic symptoms of the disease. For example, the performed
entertoxin present in food mediates the food poisoning caused by S.aureus and
Bacillus cereus.The symptoms caused by performed toxin occur much sooner than
for other forms of gastroenteritis because the effect is like eating a food poison and
the bacteria do need to grow for the symptoms to occur. Most exotoxins are rapidly
inactivated by moderate heating (60ºC), notable exceptions being staphylococcal
enterotoxin and E.coli heat stable toxin. In addition, traetment with dilute
formaldehyde destroys the toxic activity of most exotoxins, but does not affect their
antigenicity. Formaldehyde-inactivated toxins, called toxoids, are thus useful in
preparing vaccines.
IMMUNITY
The term immunity refers to all mechanisms used by the body as protection
against enviromental agents that are foreign to the body. Theses agents may be
microorganisms or their products, chemicals, drugs, pollen, or animal hair and dander
43
INNATE IMMUNITY
1. Reflex:
a. The cough and gag reflex prevent particles from entering the lung.
b. Sneezing works to expel explosively infectious agents from the nasal pasages.
c. Swallowing moves potential pathogens into the stomach, where they are
destroyed by stomach acidity.
2. Intact skin:
a. Sebaceous gland secretions may be inhibitory.
b. A dry surface is not conducive to growth of microbes.
c. Continuously sloughing epithelial cells carry adherent microbes away.
d. Sweat removes microorganisms through a flushing action and contains inhibitory
substances, such as lysozyme.
3. Conjuctive:
a. The flushing action of blinking and tears prevents colonization
b. Lysozyme and other antibacterial substances in tears are nonspecific effectors.
Lysozyme hydrolyzes structural polysaccharide in bacterial cell wall.
6. Urinary tract:
a. Flushing of urine periodically prevents bacterial population buildup.
b. Acidity of urine is inhibitory to some organisms.
c. Prostatic secretions containing spermine and zinc, which inhibit growth of some
bacteria, are introduced into the urine in males.
7. Female vagina:
a. Vaginal epithelium is sloughed rapidly and carries with it transient microbe.
b. The secretions of the vaginal tract are acidic and contain antimicrobial
substances; they also promote flushing of microorganisms from the body.
Intracellular killing. There are two principal mechanisms whereby phagocytic cells
kill internalized bacteria:
1. oxygen-dependent killing: toxic oxygen radicals (hydrogen peroxide, superoxide
ions) produced by the phagocyte cell membrane are secreted into the phagosome;
45
2. lysosomal killing: the phagosome fuses with lysosomes (granules in the cytoplasm)
containing enzymes that degrade the engulfed organism.
Fig. 15 Phagocytosis
2. Interferone-α (IF- α) proteins are released by cells once they have been infected
by a virus. The interferones protect neighboring cells from viral multiplication by
inducing the production of intracellular antiviral proteins. Interferone-α also enhances
killer Cell activity.
46
Inflammation
ACQUIRED IMMUNITY
The acquired immune response has several generalized features that characterize it
and serve to distinguish it from other physiologic systems such as circulation,
respiration, reproduction. These features are:
1. Foreignness
Animals normally do not respond immunologically to “self”. Thus, for example, if
a rabbit is injected with it is own serum albumin, it will not mount an immune
response; it recognizes the albumin as self. In contrast, if rabbit serum albumin is
injected into a guinea pig, the guinea pig recognizes the rabbit serum albumin as
“foreign” and mounts an immune response against it.
In general, compounds that are part of self are not immunogenic to the individual.
However, there are exceptional cases in which an individual mounts an immune
response against his or her own tissues. This condition is termed autoimmunity.
3. Chemical Complexity
In general, an increase in the chemical complexity of a compound is accompained
by an increase in it is immunogenecity.
Antigenicity
2. Lipids. Lipids are rarely immunogenic, but an immune response to lipids may be
induced if the lipids are conjugated to protein carriers. Thus, in a sense, lipids may be
regarded as haptens.
3. Nucleic acids are poor immunogens by themselves, but they become immunogenic
when they are conjugated to protein carriers.
4. Proteins. Virtually all proteins are immunogenic. Thus, the most common
immune responses are those to proteins. In general, proteins are multideterminant
antigens.
Immunologic Adjuvants
To enhance the immune response to a given immunogen, various additives or
vehicles are often used. An adjuvant is a substances that, when mixed with an
immunogens, enhance the immune response against the immunogen. A hapten will
become immunogenic when conjugated convalently to a carrier; it will not become
immunogenic if mixed with an adjuvant. Thus, an adjuvant enhance the immune
response to immunogens but dose not confer immunogenicity to haptens.
During artificial immunization (e.g., vaccines), and adjuvant is used to enhance the
response to antigen. Adjuvants usually prolong the presence of antigen in tissue and
activate or promote uptake of the immunogen by macrophages and lymphocytes.
There are various widely used adjuvants. One such adjuvant is Freund’s complete
adjuvant, which consists of a water-in-oil emulsion and killed Mycobacterium
tuberculosis or Mycobacterium butyricum.
ANTIBODY STRUCTURE
One of the major functions of the immune system is the production of soluble
proteins that circulate freely and exhibit properties that contribute specifically to
immunity and protection against foreign material. These soluble proteins are the
antibodies, which belong to the class of proteins called globulins because of their
blobular structure. Today they are known collectively as immunoglobulins (Ig).
molecule. The COOH-terminal domains (CH1, hinge, CH2, CH3, CH4) have been
collectively defined as the constant region, because the polypeptide backbone is
generally invariant within a particular class of immunoglobulin.
Proteolytic treatment with the enzyme papain split the immunoglobuli molecule
into three fragments of about equal size (Fig. 8). Two of these fragments were found
to retain the antibody’s ability to bind antigen specifically. These two fragments are
referred to as Fab (fragment antigen binding) fragments. The third fragment is called
Fc (fragment-crystallizable). It cannot bind the antigen, but it is responsible for the
biological functions of the antibody molecule after antigen has been bound to the Fab
part of the intact molecule.
1. Ig G
In healthy adults, constitutes approximately 85% of the total serum
immunoglobulins in adults. Ig G is approximately equally distributed between
intravascular and extravascular serum pools. One important biological function of Ig
G it is unique ability to cross the placenta, which affords protection for the fetus amd
newborn. Human Ig G has been divided into four subclasses on the basis of unique
antigenic determinants. Relative subclass percentages of the total Ig G in serum are:
Ig G1, 60 to 70%; Ig G2, 14 to 29%; Ig G3, 4 to 8%; and Ig G4, 2 to 6%.
Except for the Ig G3 subclass, which has a rapid turnover, with a half-life of 7
days, the half-life of Ig G is approximately 23 days, which is the longest half-life of
all Ig isotypes. The persistence in the serum makes Ig G the most suitable for passive
immunization by transfer of antibodies.
Ig G is the most versatile class of antibody, capable of carrying out numerous
biological functions that range from neutralization of toxin to activation of
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2. Ig M
Ig M is a pentametric immunoglobulin of about 900,000 MW that constitutes about
10% of serum immunoglobulins in healthy individuals. Ig M antibodies are clinically
significant because they predominate in early immune response to most antigens.
Along with Ig D, Ig M is a major immunoglobulin that is expressed on the surface of
B cells. Ig M antibody is present in pentametric form; of all classes of
immunoglobulin it is the best agglutinating and complement-activating antibody.
3. Ig A
Ig A is the major immunoglobulin in external secretions such as saliva, mucus,
sweat, gastric fluid, and tears. It is, moreover, the major immunoglobulin of
colostrum and milk, and it may provide the neonate with a major source of intestinal
protection against pathogens. In contrast, 10% of the circulating serum Ig A is
polymeric, while 90% is monomeric. Together, they constitute approximately 15% of
the total serum immunoglobulins. Two subclasses of Ig A have been identified (Ig A1
and Ig A2). Ig A, has a half-life of 5.5 days
In terms of complement activation, Ig A poorly activates the classical pathway. In
contrast, Ig A reportedly activates the alternative pathway of complement to provide
some direct protective functions. Ig A, once bound to bacterial or parasitic surface
antigen, may bind receptors on inflammatory cells, leading to their destruction by
means of antibody-dependent cell-mediated cytotoxicity. Moreover, its binding to
viral or microbial surface antigens may restrict the mobility of microorganisms and
prevent their binding onto mucosal epithelium.
4. Ig E
Ig E (190,000 MW) exists in serum in a monomeric form. It is also termed reaginic
antibody, has a half-life in serum of 2 days, the shortest half-life of all classes of
immunoglobulins. While Ig E constitutes only 0.004% of the total serum
immunoglobulins, is of paramount importance in hypersensitivity reactions. It also
appears to be of importance in protection against parasitic infections. The Fc portion
of Ig E binds with high affinity to receptors on mast cells and, on contact with
antigen, it triggers the degranulation of mast cells, resulting in the release of
pharmacologically active substances that mediate the hypersensitivity reactions.
5. Ig D
Ig D is a monomer of approximately 180,000 MW. While Ig D is normally
present in serum in small amounts (0.2% of total serum immunoglobulins), it is
predominantly found with Ig M on the surface of human B lymphocytes. Despite
suggestions that Ig D may be involved in B-cell differentiation, its principal function
ia as yet unknown. Serum Ig D has a half-life of 2.8 days.
52
Primary Response. In the primary response, the first class of antibody detected is
generally Ig M, which in some instances may be the only class of immunoglobulin
that is made. If production of Ig G antibody ensues, its appearance is generally
accompained by a rapid cessation of production of Ig M.
Hematopoietic bone marrow stem cells give rise to all blood cells, including the
lymphoid progenitor cells. Lymphocyte maturation, differentiation, and proliferation
take place in the lymphatic organs. Theses include the primary lymphoid tissues and
organs (The thymus gland in which T lymphocytes develop, and the bone marrow, in
which B lymphocytes develop), and the secondary lymphoid tissues and organs (of
which the spleen and lymph nodes are the most important), where mature
lymphocytes encounter and respond to foreign antigens (Fig 19).
pouches (Fig. 20). During fetal development, the size of the thymus increases. The
growth continues until puberty. Thereafter, the thymus undergoes atrophy with aging.
The thymus is a lymphoepithelial organ and consists of epithelial cells organized
into cortical and medullary areas that are infiltrated with lymphoid cells
(thymocytes). The cortex is densely populated with lymphocytes of various sizes,
most of which are immature. T lymphocytes mature in the cortex and migrate to the
medulla, which they then leave to enter the peripheral blood circulation, through
which they are transported to the secondary lymphoid organs. Thymocytes
differentiate in the thymus into subpopulations characterized by specific surface
glycoproteins such as CD3, plus CD4 and CD8. Maturation of the T lymphocytes
involves the commitment of a given determinant or epitope of a foreign antigen. This
recognition is achieved by a specific receptor on the T cell, wich is acquired during
differentiation in the thymus.
1. Spleen
This organ is highly effective at removing foreign substances and damaged blood
cell elements (for example, platelets and aged red blood cells) present in the
circulation. The spleen thus serves as the “oil filter” of the circulation. It is also the
site where many of antibodies directed against foreign substances are synthesized.
The spleen is organized into red pulp and white pulp, the latter being primarily found
surrounding small arterioles (Fig. 21). White pulp is very rich in lymphoid cells,
approximately 50% of the lymphocytes consising of mature B cells located in the
follicles, and 35% consisting of mature T cells located in the periarteriolar sheet.; 30-
40% are T lymphocytes. The red pulp is a storage site for blood cells and the site of
turnover of aged plateletes and erythrocytes.
Fig. 21 Spleen
2. Lymph Nodes
These structures are located in numerous regions throughout the body. They are like
“mini oil filters” that clear material from the tissues that drain to the lymph nodes. A
lymph node consists of the following three layers (Fig. 22):
- The cortex, the outer layer that contains mainly B cells and macrophages arranged
in clusters called follicles.
- The paracortex, which contains dendritic cells that bring antigens from the tissues to
be presented to the T cells to initiate immune response.
- The medulla, which contains B and T cells and antibody-producing plasma cells.
All of the cells involved in host defense and immunity (Fig. 21) are derived during
hematopoiesis from stem cells in the bone marrow. It is generally accepted that the
lymphoid, monocyte/myeloid, and megakaryotic lines of leukocytes diverge and
differentiate from a common, pluripotential stem cell.
and concentrate at the site of infection in response to chemotactic factors. They ingest
bacteria by phagocytosis and expose the bacteria to antibacterial substances and
enzymes contained in primary (azurophilic) and secondary (specific) granules.
Azurophilic granules are contains myeloperoxidase, ß-glucoronidase, elastase, and
cathepsin G. Specific granules serve as reservoirs for lysozyme and lactoferrin.
Eosinophils are heavely granulated cells (11 to 15 µm in diameter) with a bilobed
nucleus that stain with the acidic dye eosin. They are also phagocytic, motile, and
granulated. The granules contain acid phosphatase, peroxidase, and eosinophilic basic
proteins. Eosinophils play a role in the defense against parasitic infections. The
eosinophilic basic proteins are toxic to many parasites.
Basophils, another type of granulocyte, are not phagocytic but release the contents
of their granules during allerging responses (type 1
hypersensitivity).
Fig. 25 Monocytes
Macrophages (large eaters) are long-lived cells that are phagocytic, contain
lysosomes and, unlike neutrophils, have mitochondria (Fig. 24). Macrophages have
the following basic functions: (1) phagocytosis, (2) antigen presentation to T cells to
initiate specifi immune response, and (3) secretion of cytokines to activate and
promote innate and immune responses.
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Fig. 26 Macrophage
Macrophages express cell surface receptors for the Fc portion of IgG and for the
C3b product of the complement cascade. These receptors facilitate the phagocytosis
of antigen, bacteria, or viruses coated with these proteins. Macrophages also express
the class II MHC antigens (major histocompatibility complex), which allows these
cells to present antigen to CD4 helper T cells to initiate the immune response.
Macrophages secret interleukin-1, interleukin-6, interleukin-12, and tumor necrosis
factor in response to bacterial interaction, which stimulate immune and inflammatory
responses, including fever. A T-cell-derived lymphkine, interfero-γ, activates
macrophages and enhances their phagocytic, killing, and antigen-presenting
capabilities.
Dendritic cells of spleen and lymph nodes, interdigitating cells of the thymus, as
well as Langerhans cells in the skin probably also belong to this lineage of cells.
These cells are not very phagocytic but they are potent antigen-presenting cells.
Lymphocytes
The lymphocytes are 6 to 10 µm in diameter, smaller than leukocytes. The two
major classes of lymphocytes, B cells and T cells, have a large nucleus and smaller,
agranular cytoplasm. Although B and T cells are indistinguishable by morphologic
features, they can be distinguished on the basis of function and surface markers.
Lymphoid cells that are not B and T cells are large granular lymphocytes also known
as natural killer (NK) cells.
B cells: The primary function of B cells is to make antibody, but they also
internalize antigen and present the antigen to T cells to initiate or enhance the
immune response. B cells can be identified by by the presence on their cell surfaces
of immunoglobulins (Ig M and Ig D), class II MHC molecules, and receptors for the
C3b and C3d products of the complement cascade.
The B cell name is derived from its site of differentiation in birds, the bursa of
Fabricius and the bone marrow of mammals. B cell differentiation also takes place in
the fetal liver and fetal spleen. Activation of B cell leads to proliferation and
differentiation into a plsma cells which have small nuclei and large cytoplasm, are
factories for antibody production.
T cells: Acquired their name because they develop in the thymus. T cells have the
following two major functions in response to foreign antigen:
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2. Directly kill virally infected cells, foreign cells(e.g., tissue grafts), and tumors.
T cells were initially distinguished from B cells on the basis of their ability to bind
and surround themselves (forming rosettes) with sheep erythrocytes through the CD2
molecule. All T cells express an antigene-binding T-cell receptor (TCR), which
resembles but differs from antibody, and CD2 and CD3-associated proteins on their
cell surface. The CD3 complex is the signal transduction unit for the TCR.
There are two important types of T cells:
1. Helper T cells (CD4 T cells), wich serve as helper cells for other lymphocytes
(B cells or CD8 T cells), phagocytes, and natural killer cells.
2. Cytotoxic T cells (CD8 T cells), which specialize and identifying and killing
tumor cells, and cells infected with viruses.
According to antigenic determinants and function, T cells have been divided into
subtypes of cells including:
B. Effector T Cells
1. T-cytotoxic cells (TC). Tc cells are important for eliminating virally infected
cells, foreign tissue tissue transplants, tumor cells . They have CD8 molecule interact
with cells that bear MHC-class I molecule on their outer membrane.
2. T-delayed-type hypersensitivity (TDTH). These cells (Th1 cells) releases
cytokines that induce the migration and activation of monocytes and macrophages,
leading to the so-called delayed-type hypersensitivity inflammatory reactions.
Natural Killer Cells (NK): These cells are large, nonphagocytic, granular
lymphocytes that destroy abnormal host cells, such as those that are virus-infected or
neoplastic. They are do not display a unique antigen specificity, but when activated
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with antibodies they became effector cells against appropriate target cell antigens.
Target cell killing is accomplished by NK cell release of compounds that have a
variety of toxic effects. The cytotoxic actions of NK cells are stimulated bu
interferons and interleukine-2. Cytotoxic T cells that participate in the adaptive
immune response are also characterized as killer cells. They apear later than the NK
cells, at a time when the adaptive response has been mounted. Cytotoxic T cells kill
target cells using mechanisms identical to those used by NK cells.
CYTOKINES
Interleukin-4 Th cells (Th2) and mast cells Growth factor for B cells and Th2; inhibits
(IL-4) Th1 cells
Interleukin-6 T cells and many others T-cell activation and IL-2 production;
(IL-6) Stimulates B-cell Ig production
Interleukin-7 Fibroblasts, endothelial and Growth factor for Per-T and Per-B cells
(IL-7) some T cells
(IL-8)
Colony stimulating factor T cells and monocytes Growth and differentiation of hema.cells
____________________________________________________________________
IMMUNE RESPONSE
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Humoral Immunity
The triggering of the humoral response involves the cooperation of the T helper
cells, B cells and macrophages. Th cells recognize the foreign antigen in combination
with the MHC class II molecule and through a series of events act by triggering B
cells. As a consequence of activation by antigen, Th cells secrete cytokines, soluble
factors that effect B cells and activated them.
When activated the B cells undergo several cell divisions to produce memory cells
and plasma cells, the antibody producing cells. During the primary antibody response
or the first encounter with an antigen, it takes several days for antibody to be detected
in the serum. This lag time represents the time required for processing, selection, and
transformation of enough plasma cells for production of antibody. During the
secondary antibody response, antibody appears more quickly because of the presence
of memory cells.
Antibodies of different classes and types are prouced to different microbial
antigens and to microbial products. These substances are generally important against
extracellular organisms.
The reaction between antigen and antibody serves to induce events such as:
- In combination with complement, antibodies can lyse selected bacteria and
viruses.This occurs more efficiently with gram-negative bacteria.
- Antibodies in conjugation with complement neutralize some viruses, preventing
their attachment to target cells.
- Antibodies can also neutralize the effects of various bacterial toxins and enzymes.
- Secretory Ig A antibodies prevent the adherence of bacteria and viruses to
mucosal surface and neutralize viruses.
- Antibodies also act as effective opsonins, thus enhancing phagocytosis
The helper T cells (CD4) activate and control immune and imflammatory
responses by releasing cytokines. Helper T cells interact with peptide antigens
presented on class II MHC molecules expressed on antigen-presenting (macrophages
and B cells) cells (Fig. 27).
Cytotoxic and suppressor T cells (CD8) “patrol” the body for cells expressing virus
or tumor-like abnormal proteins presented by the class I MHC molecules.
Cellular immunity, as opposed to humoral immunity, encompasses a broad
spectrum of immune phenomena mediated by cells of the immune system. The latter
comprise various subsets of lymphocytes: T, B, and NK cells. Monocytes and
granulocytes also participate in cellular immune reactions. They have the capability
to mediate non-MHC-restricted cytotoxicity and to release a variety of enzymes and
cytokines.
In fact, CMI may be manifested in different ways depending on the antigen and the
group of T cells involved. Manifestations of CMI include delayed hypersensitivity
and cytotoxicity reactions.
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ANTIGEN-ANTIBODY INTERACTIONS
No covalent bonds are involved in the interaction between antibody and an epitope.
Consequently, the binding forces are relatively weak. They consist mainly of van der
Waals forces, electrostatic forces, and hydrophobic forces, all of which require a very
clse proximity beween the interacting moieties. Thus the interaction requires a very
close fit between an epitope and the antibody, a fit that is often compared to that
between a lock and a key. Because of the low levels of energy involved in the
interaction between antigen and antibody, antigen-antibody complex can be readily
dissociated by low or high pH, by high salt concentrations, or by chaotropic ions,
65
such as cyanates, which efficiently interfere with the hydrogen bounding of water
molecules.
Agglutination Reactions
red blood cells by Ig G antibodies. The distance between the Fab arms of the Ig G
molecule, even it is most extended form, is too short to allow effective bridging
between two red blood cells across the zeta potential. Thus, although Ig G antibodies
may be directed against antigens on the charged erythrocyte, agglutination may not
occur because of the repulsion by the zeta potential. On the other hand, some of the
Fab areas of Ig M pentamers are far enough apart and can bridge red blood cells
separated by the zeta potential. This property of Ig M antibodies, together with their
pentavalence, is a major reason for their effectiviness as agglutinating antibodies.
The use of heterologous anti-immunoglobulin antibodies may bridg between
antigenic particles that are bound nonagglutinating antibodies, leading to
agglutination. This sequence of events is the basis for the coombs test.
Latex Agglutination (LA). Latex polystyrene beads were first used to detect some
antigenic factors in serum. Either antigen or Ig G antibody is nonspecifically
66
absorbed to the surface of the latex polyeteren beads. Addition of the specific
antibody or antigen visibly agglutinates the milky-white latex suspension. Although
latex agglutination tests can be done in test tubes, they are usually performed on
slides. LA is widely used for the test of pregnancy, which involves the detection of
human chorionic gonadotropin (HCG) in the urine of pregnant women, and other
factors.
Hemagglutination (HA)
Hemagglutination is a method that visualizes the antigen-antibody interaction by
using red blood cells previously coated with the antigen under investigation. Using a
variety of different methods, specific soluble antigens can be coupled to red blood
cells. Then, when the patient’s serum is added, the antibody present binds the antigen
on the cell surface, cross-links different cells together, and causes the cells to
agglutinate.
Precipitation Reactions
There are three important areas under the curve shown in Figure 27c: (1) the zone of
antibody excess, (2) the equivalence zone, and (3) the zone of antigen excess. In the
equivalence zone, the proportion of antigen to antibody is optimal for maximal
precipitation; in the zones of antibody excess or antigen excess, the proportions of the
reactants do not lead to efficient cross-linking and formation of precipitate.
Precipitation reactions between soluble antigens and antibodies can take place not
only in solution but also in semisolid media such as agar gels. When soluble antigen
and antibodies are placed in wells cut in the gel, the reactants diffuse in the gel and
form gradients of concentration, with the highest concentrations closest to the wells.
Somewhere between the two wells, the reacting antigen and antibodies will be
present at proportions that are optimal for formation of a precipitate.
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Immunoassays
Solid-Phase Immunoassay
Immunofluorescence (IF)
COMPLEMENT
The complement fixation test was used extensively in the past for the detection of
hepatitis B surface antigen, and the Wassermann test for syphilis, but now less labor-
extensive, less expensive, and more sensitive analyses are used in clinical medicine.
The complement fixation test is based on the competition, for complement,
between various antigen-antibody complexes and the lytic system consisting of red
blood cell specific antibodies and red blood cells (RBCs), which, together with
complement, bring about the lysis of RBC (Fig. 33)
The test consists of an indicator system composed of predetermined amounts of
sheep red blood cells (SRBCs), rabbit antibodies to SRBC (also termed hemolysin),
and complement ( C ), which is generally supplied as guinea pig serum.
Preincubation of that fixed amount of complement with an antigen-antibody
system (not related to SRBC and anti-SRBC) that would results in the attachment of
complement to the antibody in this antigen-antibody complex. This attachment or
fixation of complement results in the activation of the complement cascade and the
consumption of complement components by the antigen-antibody complexes.
Subsequent introduction of sensitized SRBC into the mixture does not result in lysis
of the SRBC, because complement has been fixed or used by the first antigen-
antibody system.
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IMMUNOPATHOGENESIS
Hypersensitivity Response
Among the different types of allergic reactions caused in this manner are the
folowing:
Anaphylaxis. When a specific allergen (e.g., penicillin) is injected directly in to
the circulation, it can react in widespread areas of the body with the basophils of the
blood and mast cells located immediately outside the small blood vessels if these
have sensitized by attachment of Ig E reagins. The histamine released into the
circulation causes body-wide vasodilation as well as increased permeability of the
capillaries with resultant marked loss of plasma from the circulation. Then much
more severe consequences would ensue. This could include difficulty in breathing
because of constriction of bronchiolar muscles, uterine cramps, or involuntary
urination and defecation. In addition, widw-spread vascular permeability could
produce a massive loss of fluid into tissue spaces (hives and edema) and a drastic fall
in blood pressure (shock).
Urticaria. Urticaria results from antigen entering specific skin areas (such as
mosquito saliva injected during a mosquito bite) and causing localized anaphylactoid
reactions. Histamine released locally to causes (1) vasodilation that induces an
immediate red flare and (2) increased local permeability of the capillaries that leads
to swelling of the skin in another a few minutes. The swellings are commonly called
hives.
Hay Fever. In hay fever, the allergen-Ig E reaction occurs in the nose. Histemine
released in response to the reactions causes local vascular dilitation with resultant
increased capillary pressure, as well as increased capillary permeability. Both of these
effects cause rapid fluid leakage into the tissues of the nose, and the nasal linings
become swollen and secretory.
Asthma. Asthma often occurs in the allergic type of person. In these, the allergen-
Ig E reaction occurs in the bronchioles of the lungs. Here, the most important product
released from the mast cells seems to be the slow-reacting substances of anaphylaxis,
which causes spasm of the bronchiolar smooth muscle. Consequently, the person has
73
difficulty breathing untile the reactive products of the allergic reaction have been
removed.
It is widely recognized that many allergic reactions are due to food allergens.
Various foods or their metabolic derivatives sensitize the gastrointestinal tract,
leading to the production of Ig E antibodies. Subsequent exposure to these allergens
may result in mast cell degranulation and the release of mediators that initiate
urticaria or asthmatic disorders.
Autoimmune Response
IMMUNODEFICIENCY
Immunosuppression
VACCINES
The term vaccine is derived from vaccinia virus, a less virulent member of the
poxvirus family that was used to immunize people against smallpox. Vaccines can be
subdivided into two groups on the basis of whether they infect the person (live
vaccines) or not (inactivated vaccines).
Live Vaccines
Live vaccines are prepared with organisms limited in their ability to cause disease
(avirulent or attenuated). Live vaccines are specially useful for protection against
infections caused by enveloped viruses, which require T-cell immune responses for
resolution of the infection. Immunization with a live vaccine resembles the natural
infection. Immunity is generally long-lived and, depending on the rout of
administration, can mimic the normal immune response to the infecting agent.
However, there are two problems with live vaccines, as follows:
1. The vaccine virus may still be dangerous for immunosuppressed people or
pregnant women, who do not have the immunologic resources to resolve even a
weakened virus infection.
2. The vaccine may revert to a virulent viral form.
Live bacterial vaccines include the orally administered live, attenuated Salmonella
typhi strain Ty2la vaccine for typhoid; The BCG for tuberculosis, which consists of
an attenuated strain of Mycobacterium bovis; and an attenuated tularemia vaccine.
Live virus vaccines include polio vaccine (Sabin); measles, mumps, rubella
(administered together as the MMR) and now varicella zoster have been developed.
Inactivated Vaccines
Inactivated vaccines provide a large amount of antigen to produce a protective
antibody response without the risk of infection by the agent. Inactivated vaccines can
be produced through the chemical (e.g. formalin) or heat inactivation of bacteria,
bacterial toxins, or viruses or through the purification of the components or subunits
76
of the infectious agents. These vaccines are usually administered with an adjuvant
which boosts their immunocenicity.
Inactivated rather than live vaccines are used to confer protection against most
bacteria and viruses that cannot be attenuated, may cause reccurrent infection, or
have oncogenic potential. Inactivated vaccines are generally safe, except in people
who have allergic reactions to vaccine components. For example, many vaccines are
produced in eggs and so cannot be administered to people who are allergic to eggs.
The immune response evoked by inactivated vaccines is predominantly humoral
response and is more limited than that evoked by live vaccines. The disadvantages of
inactivated vaccines in comparison with live vaccines are as following:
1. Immunity is not usually lifelong.
2. Immunity may be only humoral and not cell-mediated.
3. The vaccine dose not elicit a local IgA response.
4. Booster shots are required
5. Large doses must be used
There are three major types of inactivated bacterial vaccines: toxoid, killed
bacteria, and capsular or protein subunits of the bacteria. Inactivated viral vaccines
are available for polio, hepatitis A, influenza, rabies, and other viruses.
Subunit vaccine: Instaed of using whole organisms, vaccines can be composed of
surface structures of bacteria and the viral attachment proteins.
Vaccines against bacterial and viral pathogens are listed in the Tables and
respectively.
Immunization Programs
Infants are immunized with diphtheria, tetanus, pertussis, (DTP), and Hib
inactivated vaccines as well as the inactivated or live oral polio vaccine. The
inactivated hepatitis A vaccine can also be administered on this schdule or to adults at
risk of infection. The hepatitis B vaccine is suggested for the first year of life as well
as later in life. The live MMR and varicella-zoster vaccines are administered at 2
years of age, after the baby’s immune response has matured and maternal antibodies
have dissipated. Booster immunizations of inactivated vaccines and the live measles
vaccine are required later in life.
Adults should be immunized with vaccines for S.pneumoniae, influenza, rabies,
hepatitis B virus, and other diseases, depending on their jobs, the type of traveling
they do, and other risk factors that may make them particularly susceptible to specific
infectious agents.
78
CLINICAL BACTERIOLOGY
Classification of Bacteria
For descriptive purposes bacteria are often grouped by four main characteristics:
the Gram reaction, shape, atmospheric requirements for respiration and the presence
of spores (phenotypic classification).
Analysis of the analytic characteristic of bacteria has also been used to classify
bacteria at the genus, species, or subspecies level (analytic classification). The
chromatographic pattern of cell wall mycolic acid is unique for many of individual
species of mycobacteria and has been used for more than 25 years to identify the
most commonly isolated species.
The most precise method for classifying bacteria is by analysis of their genetic
material (genotypic classification). Organisms were initially classified by the ratio of
guanine to cytosine.
STAPHYLOCOCCUS
The name Staphylococcus is derived from the Greek term staphyl, meaning “a
bunch of grapes.” This name refers to the fact that the cells of these gram-positive
cocci grow in a pattern resembling a cluster of grapes; however, organisms in clinical
material may also appear as single cells, pairs, or short chains. Most staphylococci
are 0.5 to 1 µm in diameter and are nonmotile, aerobic or facultative anaerobic, and
catalase-positive and grow in a medium containing 10% sodium chloride and at a
temperature ranging from 18ºC to 40ºC.
The organisms are present on the skin and mucouse membranes of humans and
other mammals, and birds. Staphylococcus is an important pathogen in humans,
causing a wide spectrum of life-threatening systemic diseases; infections of the skin,
soft tissues, bones, and urinary tract; and apportunistin infections. The species most
commonly associated with human diseases are S.aureus (the most virulent and best-
known member of the genus), S.epidermidis, S.saprophyticus, and S.haemolyticus.
S.aureus colonies are golden as the result of the carotenoid pigments that form
during their growth, hence the species name. It is also the only species found in
humans that produces the enzyme coagulase; thus, all other species are commonly
referred to as coagulase-negative staphylococci.
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Staphylococcus aureus
Toxins
Enzymes
1. Lipases are lipid-hydrolyzing enzymes, which are allow the organisms to invade
cutaneous and subcutaneous tissues by splitting fats and oils accumulating on the
skin. All strains of S.aureus and more than 30% of the strains of coagulase-negative
staphylococci produce several different lipases.
2. Leucocidin is an exotoksin that contributes to the survival of the organism, it
destroys polymorphonuclear leukocytes.
3. Coagulase is an enzyme produced by the organism during it is growth. The role
of coagulase in the pathogenesis is speculative, but coagulase may cause the
formation of fibrin layer around a staphylococcal abscess, thus localizing the
infection and protecting the organisms from phagocytosis.
4. Hyaluronidase is produced by over 90% of S.aureus strains. It is an enzyme
that hydrolyzes the hyaluronic acid constituent of connective tissue ground
substances and thus facilitates the spread of the organism through the tissues.
5. Staphylokinase (fibrinolysin) is produced by virtually all S.aureus strains. It
dissolves fibrin clots and thus contributes to the spread of the organism from local
sites.
6. Nuclease is the another enzyme for S.aureus. The role of this enzyme in the
pathogenesis of infection is unknown.
7. Penicillinase. More than 90% of staphylococcal isolates were susceptible to
penicillin in 1941, the year the antibiotic was first used clinically. Resistance to
penicillin quickly developed, however, primarily because the organisms could
produce penicillinase (ß-lactamase).
8. Catalase. All staphylococci produce catalase, which catalyzes the conversion of
toxic hydrogen peroxide to water and oxygen. Hydrogen peroxide can accumulate
during bacterial metabolism or after phagocytosis.
Carbuncles occur when furuncles coalesce and extend to the deeper subcutaneous
tissue. Multiple sinus tracts are usually present.
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2. Scaled Skin Syndrome is a disease that occurs in infants and children 4 years of
age or under. In this syndrome the organisms release exfolitative toxin, which is
resposible for the extensive intraepidermal splitting and bullous necrosis of the tissue.
4. Food Poisoning is the one of the most common food poisoning in the world.
The organisms are usually introduced into food, such as processed meats, pastries,
potato salad and ice cream. The contaminated foods is kept at room temperature,
during which time the organisms multiply and release heat stable enterotoxin A or D.
Following ingestion of the food, the onset of disease is abrupt and rapid with an
incubation period of only 1-6 h. Staphylococcal food poisoning is characterized by
severe vomiting, diarrhea, and abdominal pain. The absence of fever is an important
observation in the differential diagnosis of staphylococcal food poisoning.
Laboratory Diagnosis
Specimens obtained depend on the disease process and include lesion material,
pus, sputum, blood, spinal fluid, and feces.
İsolation and identification of S.aureus requires initial cultivation on blood agar
and/or specific medium. Overnight incubation under aerobic conditions at 37ºC. The
organism may be identified as a gram-positive, catalase positive coccus exhibiting
coagulase.
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Treatment
Coagulase-Negative Staphylococci
STREPTOCOCCUS
A. Antigenic structure
Capsule. The outermost layer of the cell is the capsule, which is composed of
hyaluronic acid, identical to that found in connective tissue. For this reason the
capsule is nonimmunogenic (again in contrast with S. pneumoniae).
C-carbonhydrates are cell wall polysaccharides whose antigenic diversity forms
the basis for the classification of streptococci into 20 serogroups lettered from A to V.
Lipoteicoic acid (LTA). The ability of group A streptococci to bind to epithelial
cells in the mouth and on the skin is mediated by lipoteicoic acid exposed on the cell
surface.
M protein is a major antigen associated with virulent streptococci. In the abscence
of M protein the strains are not infectious. The M protein also prevents interaction
with complement.
Protein F (fibronectin-binding protein) mediates attachment to fibronectin in the
pharyngeal epithelium.
B. Extracellular products
Acute rheumatic fever occurs most commonly among young children during the fall
and winter, and can occur only when preceded by pharyngitis caused by any of group
A streptococcal serotypes. The disease can occur in 0.1-3% of untreated patients from
85
Laboratory Diagnosis
The specimens obtained depend on the disease process and include nose and throat
swabs, lesion material, pus, sputum, blood for culture and immunoserology, urine,
and spinal fluid.
For isolation and identification of group A streptococci initial cultivation on blood
agar or specialized selective agar are required. Overnight incubation anaerobically or
under aerobic conditions in the presence of 10% carbon dioxide at 37ºC is optimal for
isolation of the organism. The organism may be identified as beta-hemolytic, gram
positive, catalese negative coccus, and is inhibited by bacitracin.
Immunoserologic test. Both the anti-streptolysin O (ASO) and anti-Dnase B assays
are useful in diagnosis.
GROUP B STREPTOCOCCİ
Group B streptococci (S. agalactiae) are harbored in the female genital tract and
male urethra of 15-25% of humans and animals, as well as in the pharynx and GI
tract. The organism is transmitted from an infected mother to her infant in utero or at
birth. Group B streptococci are encapsulated. These organisms far outnumber E.coli
K1 as the leading cause of neonatal meningitis during the first 4 months of life. The
antiphagocytic properties of the capsular polysaccharide allow the organisms to
survivei multiply, invade epithelial cells, and induce an acute inflammatory response.
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Streptococcus pneumoniae
Disease of S. pneumoniae
Laboratory Diagnosis
Bacteria
1. Streptococcus pneumoniae 2. Haemophilus influenzae
3. Staphylococcus aureus
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Viruses
1. Influenza virus Types A and B 2. Parainfluenza viruses
3. Respiratory syncytial virus
ENTEROCOCCI
CORYNEBACTERIA
C. diphtheriae
Diseases of C. diphtheriae
sometimes covered with a greyish membrane. The ulcer may also be superinfected
with S. aureus or group A Streptococcus
Laboratory Diagnosis
Specimens obtained depend on the disease process and include a nose, throat,
nasopharyngeal, and wound swab.
Specimens are cultivate on cysteine-tellurite agar and Loeffler’s coagulated serum.
In addition, differential diagnosis necessitates primary cultivation on blood and
chocolate agar. Identification of gray-black colonies on cystein-tellurite agar, the
typical Chinese letter, beaded, barred, or palisading arrangment of pleomorphic rods
with accentuated metachromatic granules by methylene blue staining of colonies on
Loeffler’s coagulated serum, constitute presumptive evidence for C. diphtheriae.
Definitive identification of the organism is based upon the demonstration of
exotoxin pruduction by a virulence test in guinea pigs or by a modified in vitro
“Elek” test utilizing specific antitoxin.
Treatment
Diphtheroides
Diphtheroides are corynebacteria that occupy the skin, nose, throat, nasopharynx,
urinary tract, and conjunctiva of normal individuals can, in rare instances, infect
immunosuppressed hospitalized individuals, and produced septicemia with a high
cases fatality rate.
BACILLUS
Species of the genus Bacillus are gram-positive, form endospores, and are either
strictly or facultative aerobic. Most of the seventy or so species of Bacillus are found
in soil and water, and are usually encountered in the medical laboratory as airborne
contaminants. Bacillus anthracis, the organism responsible for anthrax, is the most
important member of this genus. This species is considered one of the most feared
agents of biological warfare.
Bacillus anthracis
Bacillus anthracis, has an illustrious place in medical history. It was the first
bacterium shown to be the causative agent of an infectious disease by Koch in 1877.
B.anthracis is a gram-positive, spore-forming, encapsulated, nonmotile rod with
characteristic square cut ends. The organism occurs singly, in pairs, or in long chains
with a “string of pearls” appearance. Spores are oval, centrally located and can
survive in soil for years. The organism is facultative anaerobe that grows best
aerobically on blood agar at 37ºC and is nonhemolytic. Colonies appear rough with
an irregular edge that gives a “Medusa head” appearance.
Pathogenesis
Diseases of B. anthracis
Cutaneous anthrax is the result of infection with spores that gain access to
humans through small abrasions or scratches in the skin while handling diseased
animals or animal products, such as meats, hides, shaving brushes made from
infected animals. Approximately 95% of anthrax infections are due to the inoculation
of Bacillus spores through exposed skin surfaces. This disease characterized by the
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Laboratory Diagnosis
Specimens obtained depend on the disease process and include material from
lesions, blood for culture, spinal fluid, and sputum. Gram stain of the specimen will
reveal characteristic gram-positive rods with square cut ends and often in pairs or
long chains; spores appear as an oval unstained “space” in the center of the organism.
Primary isolation and identification procedures require initial cultivation on blood
agar. Overnight incubation under aerobic conditions at 37ºC is optimal for isolation
of the organism, which is identified as a nonhemolytic, gram-positive, nonmotile,
spore-forming rod with square cut ends and occuring singly, in pairs, or in long
chains. A direct immunofluorescence assay on the isolated organism or a virulence
test in animals used for definitive identification.
Traetment
Bacillus cereus
Bacillus cereus exists as a saprophyte in water and soil. The organism differs from
B. anthracis in that it is motile, nonencapsulated, and beta-hemolytic. The organism
may cause a self-limiting type of food poisoning, usually resulting from the imgestion
of contaminated rice or meat dishes containing enterotoxin.
LISTERIA
The genus Listeria consists of seven species, with Listeria monocytogenes the only
human pathogen. L.monocytogenes is a short, gram-positive, facultatively anaerobic
bacillus. The short bacilli appear singly, in pairs, or in short chains and can be
mistaken for S. pneumoniae or Enterococcus. The organism are motile at room
temperature but not at 37ºC, and they exhibit a characteristic tumbling motion. Grows
best aerobically or under microaerophilic conditions (3 to 5% oxygen) on blood agar
at 37ºC. The organism produces a narrow zone of beta hemolysis.
Although listeria are widely distributed in nature, human disease due to this
organism is uncommon and is restricted to several well-defined populations:
neonates, the elderly, pregnant women, and patients with defective cell-mediated
immunity.
Pathogenesis
Diseases of L.monocytogenes
Laboratory Diagnosis. Specimens (blood and spinal fluid) that obtained from
patients are cultivate on blood agar and identified according to the charasteristic
features discoused above.
NEISSERIA
Neisseria gonorrhoeae
The outer surface is not covered with a true carbohydrate capsule, as is found in
Neisseria meningitidis.
Pili mediate attachment of the organism to epithelial and mucosal cell surfaces and
are antiphagocytic.
Three outer membrane proteins (OMPs) have been studied extensively:
1. Por proteins are porin proteins that form pores oe channels in the outer
membrane.
2. Opa (opacity) proteins are a family of membrane proteins that mediate binding
to epithelial cells.
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Diseases of N. gonorrhoeae
Laboratory Diagnosis
Specimens obtained depend on the disease process and include urethral, cervical,
rectal, pharyngeal, and/or conjunctival exudates.
The direct demonstration of gram-negative intracellular diplococci within PMNs is
diagnostic only when observed in the urethral exudates of males with characteristic
clinical manifestations.
Gram stains of smears from female urethral and cervical exudates, from rectal,
pharyngeal, and conjunctival exudates of male and females, are unreliable due to the
potential presence of nonpathogens resembling gonococcal morphology, and presence
of meningococcuses. All such specimens must be cultured and the isolated organism
identified. These techniques require initial cultivation on Thayer-Martin medium.
Incubation for 48 h at 35-37ºC under aerobic conditions in the presence of 3-10%
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carbon dioxide is optimal for isolation of the organism, which may be identified as a
gram-negative, oxidase positive, diplococcus that ferments glucose, but not maltose,
sucrose, or lactose.
Treatment
Neisseria meningitidis
Laboratory Diagnosis
Treatment: Early treatment with penicillin has reduced the case fatality rate in
disseminated disease from 40-90% to 10-15%, but the antibiotic is ineffective in
eradicating the carrier state. Chloramphenicol and ceftriaxone are effective in
penicillin-allergic individuals.
Polysaccharide vaccines conjugated with protein carriers offer protection for
infants younger than 2 years.
Moraxella
Members of the genus Moraxella are nonmotile, gram-negative cocobacilli that are
generally found in pairs. They are related to the neisseriae, and the two organisms can
be confused on Gram stain. Moraxella are aerobic, oxidase-positive, fastidious
organisms that do not ferment carbohydrates. The most important pathogen in the
genus is Moraxella (formerly Branhamella) catarrhalis. This organism can cause
infections of the respiratory system, middle ear, eye, CNS, and joints.
These organisms can be cultured on blood or chocolate agar, and identified by a
battery of biochemical tests.
Acinetobacter
Members of the genus Acinetobacter are nonmotile cocobacilli that are frequently
confused with neisseriae in gram-stained samples. They are generally encapsulated,
oxidase negative, obligately aerobic, and they do not ferment carbohydrates.
Acinetobacter are widely distributed in nature and are capable of infecting virtually
any body site, organ system, or tissue.
ENTEROBACTERIACEAE
1. Do not possess the cytochrome oxidase enzyme and are thus “oxidase negative”.
2. Possess enzymes that enable them to reduced nitrate to nitrite.
3. Are able to ferment glucose.
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2. Biochemical reactions.
a. Carbohydrates important for identifying Enterobacteriaceae include glucose,
lactose, sucrose, and mannitol.
b. Tests for the presence of enzymes, such as the “indol test” for tryptophanase,
the urease test, and tests for the presence of dehydrogenase and dihydrolase enzymes.
c. Methyl red, Voges Proskauer (MRVP) test for end products of glucose
metabolism.
d. The ability of an organism to produce hydrogen sulfide (H2S) gas and motility
are also important differentiating characteristics.
Pathogenesis
Numerous virulence factors have been identified in the members of the family
Enterobacteriaceae. Some are common to all genera, and others are unique to specific
virulent strains.
• Endotoxin is a virulence factor shared among all aerobic and some anaerobic gram-
negative bacteria.
• Capsule. Encapsulated Enterobacteriaceae are protected from phagocytosis by the
hydrophilic capsular antigens, which repel the hydrophobic phagocytic cell surface.
• Antigenic phase variation. Capsular and flagellar antigens can be alternately
expressed or nor expressed, a feature that protects the bacteria from antibody-
mediated cell death.
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Escherichia coli
The genus Escherichia consists of at least five species, with Escherichia coli the
most frequently isolated. E.coli is present in the GI tract in large numbers and is the
Enterobacteriaceae most frequently associated with bacterial sepsis, neonatal
meningitis, infections of the urinary tract, and gastroenteritis in travelers to countries
with poor hygiene.
Urinary Tract Infections. E. coli is responsible for more than 80% of all
community-acquired urinary tract infections
Neonatal Meningitis. E. coli, together with group B streptococci, are the most
common causes of neonatal meningitis; 75% of these Escherichia strains possess the
K1 capsular antigen.
Septicemia. Typically, septicemia caused by gram-negative bacilli such as E.coli
originates from infections in the urinary or gastrointestinal tract.
Gastroenteritis. Strains of E. coli that cause gastroenteritis are subdivided into at
least four groups:
2. Enteroinvasive E. coli (EIEC) are able to invade and destroy the colonic
epithelium, producing a disease characterized by fever and cramps, with blood and
leukocytes in stool specimens (smilar to shigellosis). Disease has been associated
with specific O serotypes of E.coli, which not produced enterotoxin.
Laboratory Diagnosis
Specimens obtained depend on the disease process and include urine, bile salts,
and rectal swab obtained during sigmoidoscopy.
Specimens are inoculated onto blood agar and Mac Conkey agar. Colonies are
usually detected after 24 h of incubation and are identified based on initial
morphology and the species is verified by biochemical tests and serologing typing.
E.coli are facultative anaerobes which ferment a wide range of sugars, including
lactose, producing acid and gas. They are oxidase (-), Voges-Proskauer and citrate (-),
but produce indol from tryptophane and are methyl red positive. They are actively
motile due to the possession of flagella.
Salmonella
surfaces. It is now known that chicken eggs can become infected in the ovaries and
carry the organism internally. Consumption of raw eggs is no longer recomended.
S. typhi infections occur when food or water contaminated by infected food
handlers is ingested. Also, person-to-person spread is common. Although exposure to
Salmonella is frequent, a large inoculum (106-8 bacteria) is required for the
development of symptomatic disease.
Pathogenesis
Salmonella invade epithelial cells of the small intestine. Disease may remain
localized or become systemic, sometimes with disseminated foci. The organisms are
facultative, intracellular parasites that survive in phagocytic cells. Development of
clinical symptomes depends on : 1) infectious dose, 2) bacterial factors.
Salmonella species are protected from stomach acids and the acid pH of the
phagosome by an acid tolerance response (ATR) gene. Catalase and superoxide
dismutase are other factors that protect the bacteria from intracellular killing.
Clinical Diseases
Enteritis
Enteritis is the most common form of salmonellosis. Symptoms generally appear 6
to 48 h after consumption of the contaminated food or water, with the initial
presentation of nausea, vomiting, and nonbloody diarrhea. Elevated temperature,
abdominal cramps, myalgias, and headache are also common. Patients usually
recover after 2-3 days and disease is limited to the GI tract in most cases.
Septicemia
All Salmonella species can cause bacteremia, although infections with S.
cholerasuis, S. paratyphi, and S. typhi more commonly lead to a bacteremic phase.
Enteric Fever
S. typhi produce a febrile illness called typhoid fever. A mild form of this disease,
referred to as paratyphoid fever, is produced by S. paratyphi A, Salmonella
schottmuelleri (formerly S. paratyphi B), and Salmonella hirschfeldii (formerly S.
paratyphi C). The disease is characterized by fever and, frequently abdominal
symptoms. Nonspecific symptoms mau include chills, sweats, headache, anorexia,
weakness, sore throat, cough, and myalgias. The incubation period various from 5 to
21 days. Patients who survive typhoid fever retain a high level of immunity to second
infections.
Laboratory Diagnosis
laboratories identify the organisms to genus biochemically and may carry out limited
serologic typing assays to place the organism into a group. Salmonella is a non-
lactose fermenter, produced acid and gas (except S. typhi) from glucose, metabolizes
citrate, and produces (except S. paratyphi A) hydrogen sulphide (H2S).
Treatment: Unless patients are acutely ill with enteric fever, antibiotic treatment is
not indicated. Ciprofloxacin, amoxicillin, or chloramphenicol is used to treat
septicemia, although this is a last resort.
During the acute and early stages of typhoid, blood, bone marrow and urine
specimens are most likely to harbor the organism. (See Table below for timing of
obtaining specimens for diagnosis of typhoid fever.)
3. Oral Ty21a live (attenuated) vaccine, given in three doses of one capsule each on
alternative days.
Shigella
The organisms survive stomach acidity, pass through the small intestine, and
adhere to the mucosal epithelium of the terminal ileum and colon. Destruction of the
superficial mucosal layer, and production of mucosal ulceration is occurs. An
exotoxin (Shiga toxin) with enterotoxic and cytotoxic properties has been isolated
from S. dysenteriae and its toxigenicity may play a secondary role in development of
intestinal lesions.
Shigellae cause classic bacillary dysentery, characterized by diarrhea with blood,
mucos, and painful abdominal cramping. Abundant neutrophils, erythrocytes, and
mucos are found in the stool. Infection is generally self-limited, although antibiotic
treatment is recommended to reduce the risk of secondary spread to family members
and other contacts.
Laboratory Diagnosis
Treatment
Yersinia
The genus Yersinia includes three species of medical importance: Yersinia pestis,
Yersinia enterocolitica and Yersinia pseudotuberculosis.
Yersinia pestis
The etiologic agent of one of the devastating diseases in history was called Black
Death (epidemic plague). During a 5-year period in the middle of the Fourteenth
century, this disease claimed 25 million people—almost one fourth of the European.
Y. pestis is a gram-negative, nonmotile, ellipsoidal rod that exhibits characteristic
bipolar staining (safety pin appearance) with special polychromatic stains. The
organism is a facultative intracellular parasite and is a facultative anaerobe that grows
best aerobically on blood agar and MacConkey agar that contain bile salts at 28ºC
and is nonhemolytic.
Pathogenesis
Bubonic plague is initiated following the bite of an infected rat flea, usually on the
lower extremitites. Within a few hours, the organisms are carried to the regional
lymph nodes, usually in the groin, where rapid multiplication occurs. After 7 days of
incubation period, chills, fever and an acute regional lymphadenitis referred to as a
“bubo” are occured. In the absence of treatment patients will rapidly progress to
bacteremia, and as many as 75% will die.
Pneumonic plague is initiated by droplet nuclei from a patient with pulmonary
disease. The organisms proceed directly to the lung parenchyma where they multiply
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rapidly. Death is rapid, occurring in less than 2 days in almost all patients, treated or
untreated.
Laboratory Diagnosis
Specimens obtained depend on the disease process and include bubo aspirates,
blood for smear and culture, sputum, and cerebrospinal fluid. These specimens are
cultivate on blood agar and MacConkey’s agar. Overnight incubation under aerobic
conditions at 28ºC is optimal for isolation of the organism. Definitive identification is
made by direct immunofluorescence of the cultured organism, which is usually
available only at regional Public Health Laboratories.
Treatment
OTHER ENTEROBACTERIACEAE
Klebsiella
Members of this genus are non-motile and have a prominent capsule that is
responsible for the mucoid appearance of isolated colonies and enhanced virulence of
the organism in vivo. The most commonly isolated member of this genus is
Klebsiella pneumoniae, which as the name implies is associated with community
acquired primary lobar pneumonia. Pneumonia caused by Klebsiella is frequently
associated with necrotic destruction of alveolar spaces, with cavity formation and the
production of blood-tinged sputum. Klebsiella are also associated with wound, and
urinary tract infections.
Proteus
The genus Vibrio is composed of many species of curved bacilli, and 12 have been
implicated in human infections. V. cholerae, V. parahemolyticus, and V. vulnificus are
the most prominant.
Vibrio cholerae
Members of the genus Vibrio are short, curved, rod-shaped that differ from
Enterobacteriaceae by their positive oxidase reaction and growth on alkaline but not
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acidic media. They are rapidly motile by means of a single polar flagellum. Vibrio
species grows regularly on routine laboratory media (within temperature range from
18ºC to 37ºC), but can be enriched by incubation in alkaline broths, such as alkaline
peptone water. Special selective media, such as thiosulfate-citrate-bile salts (TCBS)
agar, are used for isolation of V. cholerae and relation vibrios from clinical
specimens.. V. cholerae can grow in the absence of salts; most other species that are
pathogenic in human require salt.
Members of the genus are subdivided on the basis of their somatic O antigens, with
more than 200 serogroups described to date. V. cholerae O1 and O139 are responsible
for causing classic cholera, which can occur in epidemics or worldwide pandemics.
V. cholerae O1 can be subdivided into the following two biotypes: El Tor and
classical (cholerae)
Vibrios can remain viable in food and water for up to 3 weeks. Shellfish harboring
V. cholerae must be boiled for at least 10 min to kill the organisms. Chlorination of
water and standard disinfectants are able to destroy the organism easily.
Pathogenesis
is the only virulence factor of V. cholerae that has been established definitively.
Large numbers of organisms are ingested in contaminated food or water. Those that
survive the gastric acidity attach, with the aid of flagella and pili, to the brush borders
of epithelial cells in the small intestine. The vibrios multiply to large numbers rapidly
on the mucosal surface and produce Cholera toxin (or chleragen), which binds to
the ganglioside receptors on the host epithelial cells. The toxin A subunit enters into
the mucosal cells, and effect a series of reactions that result in the hypersecretion of
fluids and electrolytes into the intestinal lumen. After several hours to 3-days
incubation, patients experience a sudden onset of severe watery diarrhea with
vomiting and abdominal pain. The stool specimens are colorless and odorless, free of
protein, and speckeled with mucus flecks (rice-water stool). Untreated, the death
from sever dehydration causing hypovolemic shock may occur in hours to days, and
the death rate may exceed fifty persent.
Laboratory Diagnosis
Stool is the only specimen used for diagnosis of cholera. Clear rice-water stool or
stool containing mucus is highly suspicious. If transport is required, stool is place into
Cary-Blair or alkaline peptone water.
Stools should be inoculated into or onto standard laboratory media and TCBS
selective agar. All colonies should be screened fo indophenol oxidase enzyme.
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Treatment
1. Emphasize proper hygiene practices, such as sewage disposal and treatment, using
separate water sources for drinking and other activities.
2. Chlorine all water supplies.
3. Boil shellfish fo a minimum of 10 min before eating. Do not eat raw shellfish.
4. Vaccins are not recommended at this time because of their limited effectiveness.
No countries currently require visitors to show evidence of cholera vaccination.
Like V. cholerae, the other pathogenic vibrios are free-living enviromental bacteria.
Most species are saltwater inhabitants and require increased sodium chloride
concentrations for growth.
Gastroenteritis caused by V. parahaemolyticus can range from self-limiting
diarrhea to a cholera like illness.
Fatal septicemia caused by V. vulnificus, which is the most virulent of the
noncholera vibrios
Treatment
Aeromonas
Plesiomonas
Campylobacter
Helicobacter
Members of the genus Helicobacter are curved or spiral organisms. They have a
rapid, corkscrew motility due to multiple polar flagella. Helicobacter pylori, the
species of human significans, is microaerophilic, and produces urease. It causes acute
gastritis, and duodenal and gastric ulcer.
H. pylori is highly motile, and produces an abundance of urease. Helicobacters do
not ferment or oxidize carbohydrates, although they can metabolize amino acids by
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Laboratory Diagnosis
Breath Tests for Ureas. Breath tests involved administering radioactively labeled
urea by mouth. If H. pylori are present in the patient’s stomach, the urease produced
by the organism will split the urea to CO2 (radioactively labeled and exhaled) and
NH3.
B. Invasive Tests
1. A routine Gram stain with a modified counterstain using basic fuchsin insteat of
safranin to enhance the faint staining of these thin bacteria. The presence of typical
curved rods is confirmatory.
2. Biopsy tissue can be tested directly for the presence of urease.
3. Culturing the specimen for H. pylori is the third option. Ground tissue should be
inoculated onto fresh chocolate agar or Skirrow campylobacter agar, and incubated
under microaerophilic conditions (5-10% CO2) at 37ºC for up 1 week. Isolates of this
organism are oxidase and catalase positive.
Treatment
Several treatment regimens have been effective but long term results have not been
evaluated. Current therapies usually include:
1. Metronidazole + amoxicillin or Clarithromycin + amoxicillin
2. two antibiotics + bismuth subcitrate compound.
Acid-reducing drugs greatly enhance the effect of antibiotic treatment.
Pseudomonas
Pseudomonas aeruginosa
Pathogenesis
P. aeruginosa begins with attachment to and colonization of host tissue. Pili on the
bacteria mediate adherence, and a glycocalyz capsule reduces the effectiveness of
normal clearance mechanisms. Host tissue damage facilitates adherence and
colonization. P. aeruginosa produces numerous toxins and extracellular products that
promote local invasion and dissemination of the organism. These include:
Diseases of P. aeruginosa
Laboratory Diagnosis
Any infected tissue, blood urin, sputum, or other appropriate specimen may be
collected for microbiological evaluation.
Because pseudomonads have simple nutritional requirements, they grow easily on
such common isolation media as blood agar or MacConkey agar. Aerobic incubation
is required (unless nitrate is available), so growth in broth is generally confined to the
broth-air interface.
The colonial morphology (colony size, hemolytic activity, pigmentation, odor)
combined with selected rapid biochemical tests (e.g., positive oxidase reaction) are
used for the preliminary identification of isolates. P. aeruginosa grows rapidly and
has flat colonies with a spreading border, green pigmentation (caused by production
of blue pyocyanin and yellow fluorescein), and a characteristic sweet, grapelike odor.
Treatment
Acinetobacter
Acinetobacters are recovered in nature and in the hospital. They survive on moist
surfaces, including respiratory therapy equipment, and on dry surfaces such as the
human skin (the latter feature is unusual for gram-negative bacilli).
Acinetobacters are opportunistic pathogens that cause infections in the respiratory
tract, urinary tract, and wounds; they also cause septicemia. Treatment of
Acinetobacter infections is problematic because these organisms are often resistant to
antibiotics. Specific therapy must be guided by in vitro susceptibility tests.
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Moraxella catarrhalis
Bordetella
Bordetella organisms are very small (0.2 to 1.0 µm), strictly aerobic,
nonfermentative, gram-negative coccobacilli. Seven species are currently recognized,
with three species responsible for human disease: Bordetella pertussis, the agent
responsible for pertussis or whooping cough; Bordetella parapertussis, responsible
for milder form of pertussis; and Bordetella bronchiseptica, responsible for
respiratory disease in dogs, swine, laboratory animals, and occationally pertussis-like
symptoms in humans.
Bordetella pertussis
Pathogenesis
subunit binds to receptors on phagocytic cells. Two S4 subunits are present in each
toxin molecule.
B. pertussis produces several toxins that mediate the localized and systemic
manifestations of disease.
. S1 portion of pertussis toxin
. hemolysin toxin
. dermonecrotic toxin
. tracheal toxin
. lipoplysaccharide
Laboratory Diagnosis
Treatment
Francisella tularensis
Francisella tularensis is the causative agent of tularemia (also called rabbit fever)
in animals and humans. Tularemia was first isolated in 1912 from “plaguelike
disease” among ground squirrels in Tulare County, CA. The organisms are harbored
in the blood and tissues of wild and domestic animals, including rodents. In the
United States, the chief reservoir hosts are wild rabbits and ground squirrels.
Transmission is through the skin and/or conjunctiva from handling infected
animals, through the skin from the bite of infected blood-sucking deer flies and wood
ticks, through the GI tract from the ingestion of improperly cooked meat or
contaminated water, and through the respiratory tract by aerosol inhalation.
F. tularensis is a gram-negative, nonmotile, coccobacillary or pleomorphic rod.
Virulent organisms appear to be encapsulated and are facultative intracellular
parasites. Thses fastidious organisms are strict aerobes that grow best on blood-
glocose-cysteine agar.
Organisms entering through the unbroken or abraded skin, GI tract, or conjunctiva,
establish residence locally and in the regional lymph nodes. They enter macrophages
and mononuclear phagocytes with intracellular multiplication resulting in ulcer
formation, and regional lymphadenopathy accompained by fever, nausea, vomiting,
and/or abdominal pain.
BRUCELLA
Brucella is the causative agent of brucellosis (undulant fever, Malta fever). This
disease is an acute or chronic recurrent disease transmissible from animals to humans.
Although a wide range of animal reservior hosts exist for each of the four species
capable of producing human disease, they exhibit significant animal-host specificity.
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The major reservoir hosts are cattle for Brucella abortus, goats and sheep for
Brucella melitensis, swine for Brucella suis, and dogs (particularly beagles) for
Brucella canis.
Upon contact with the organisms, pregnant animals develop either an
asymptomatic infection or uterine and mammary glad disease that culminates in
abortion. Erythritol, an alcohol present in the placenta and fetal fluids of animals but
not humans, srves both as a growth factor for virulent brucellae and enhances
intracellular phagocytic growth, thereby accounting for the occurrence of fetal
predilection and abortion only in animals. The animals shed the organisms in their
milk for weeks or months during the carrier state and after recovery.
Transmission to humans is from the ingestion of contaminated raw milk or dairy
products in most parts of the world, and from the handling of infected animals by
high risk individuals, such as veterinarians, butchers, meat packers, and farmers.
Brucella gain entry through the broken or unbroken skin, by ingestion, or through
the conjunctiva. After a variable incubation period of a few days to several weeks or
months, the acute phase is ushered in and is characterized by an undulant fever,
chills, sweating, and often times fatigue. Regional lymphadenopathy,
hepatosplenomegaly, septicemia, osteomyelitis and an acute arthritis sometimes
occur.
Laboratory Diagnosis
Specimens for laboratory diagnosis depend on the disease process and include
lymph node and bone marrow aspirates and blood for culture and serology.
Brucella are slow-growing, fastidious organisms on primary isolation. Primary
isolation may be enhanced by using a biphasic blood culture system containing
trypticase soy and/or Brucella agar enriched agar incubated aerobically under 5%
carbon dioxide for minimum of 4 weeks or by using one of the newer automated
blood culture systems. Organisms are identified as Brucella on the basis of
biochemical reactions and are speciated on the basis of hydrogen sulfide production;
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growth in the presence of the dyes basic fuchsin and thionin; and agglutinin
absorption assay.
Treatment
PASTEURELLACEAE
HAEMOPHILUS
Most species of Haemophilus (from the Greek words for “blood-loving”) require
supplementation of media with growth-stimulating factors, specifically X factor
(hemin) and/or V factor (nicotinamide adenine dinucleotide, NAD). Although both
factors are present in blood-enriched media, the blood must be gently heated to
release the factors and destroy inhibitors of V factor. For this reason heated blood
agar (i.e., chocolate agar) is used for the in vitro isolation of Haemophilus spp., which
are differentiated by their requirements for X and V factors.
X factor is a heme portion of hemoglobin; required for synthesis of essential
enzymes.
V factor is a co-enzyme. Present in erythrocytes but unavailable for some
erythrocyte types, such as sheep RBCs, unless the cell membrane is lysed. Intact
horse and rabbit RBCs provide V factor.
The cell wall structure of Haemophilus is typical of other gram-negative bacilli.
Lipopolysaccharide endotoxic activity is present, and the surface of the cell is
covered with strain-and species-specific protein antigens. The surfaces of many but
not all strains of H.influenzae are covered with polysaccharide capsule, with six
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H. influenzae, colonize the upper respiratory tract in virtually all individuals with
in the first few months of life. These organisms can spread locally and cause disease
in the ears (otitis media), sinuses (sinusitis), and lower respiratory tract, but
disseminated disease is relatively uncommon. In contrast, encapsulated H. influenzae
(particularly serogroup b) is infrequently present in the upper respiratory tract but is
the most common cause of epiglottitis and pediatric meningitis. The organism is able
to penetrate through the nasopharyngeal submucosa and enter the blood stream. In the
absence of specific opsonic antibodies directed against the polysaccharide capsule,
high-grade bacteremia can develop, with dissemination to the meninges or other
distal foci.
The clinical syndromes that accompany infections of H. influenzae are :
Meningitis. H.influenzae type b is the most common cause of pediatric meningitis,
which is indistinguishable clinically from that of the other acute bacterial
maningitides. Clinical signs of the disease include; fever, vomiting, lethargy, nuchal
rigidity.
Epiglottitis is a life-threatening type of cellulitis that starts at the epiglottis and
spreads into surrounding tissues. The child will have pharyngitis, fever and breathing
difficulties, which can rapidly progress to complete obstruction of the airway and
death.
Arthritis. Infection of single large joints, secondary to bacteremic spread of H.
influenzae type b, is the most common form of arthritis seen in children less than 2
years of age.
Conjunctivitis and Brazilian Purpuric Fever. Epidemic, as well as endemic,
conjunctivitis can be caused by H. influenzae biotype aegypticus. Aspecific strain of
this organism has also been associated with Brazilian purpuric fever, a fulminant
pediatric disease characterized by fever, vomiting and abdominal pain, and then rapid
development of petechiae, purpura, shock and death.
Otitis, Sinusitis, and Lower Respiratory Tract Disease.
Laboratory Diagnosis
In cases of epiglottitis, direct culture collection from the affected throat is not
recommended, since the trauma may cause airway obstruction. Blood cultures are
often positive.
Microscopy. Gram stain is usually sensitive.
Primary Isolation and Identification. Isolation of H. influenzae from clinical
specimens is relatively easy if media supplemented with X and V factors are
inoculated. Chocolate agar is used in most laboratories, the bacteria appear as 1 to 2
mm smooth opaque colonies after 24 hours of incubation. Growth on unheated blood
agar plates can also be detected surrounding colonies of S. aureus [(satellite
phenomenon) (süt anne etkisi)]. The staphylococci provide required growth factors
by lysing the erythrocytes in the medium and secreting V factor. The size of the
colonies of H. influenzae is much smaller than on chocolate agar because the V factor
inhibitors are not inactivated.
Identification of isolates is based on requirement for X and V factors, enzymatic
and biochemical test results, hemolytic activity on rabbit blood.
Treatment
Haemophilus ducreyi
H. ducreyi the etiologic agent for chancroid, or soft chancre, was first described by
Ducrey. This sexually transmitted disease is distinguished from syphilis by the
presence of a painful, nonindurated genital ulcer with well-defined margins. The
disease has worldwide distribution, and is generally present in poor socioeconomic
areas.
Laboratory diagnosis is difficult because the organism sometimes fails to grow in
vitro and, when it does grow, incubation for 3 or more days is required. In general,
diagnosis is based on the clinical presentation of the patient and a history of
chancroid in the community.
Treatment with ceftriaxone, erythromycin, ciprofloxacin, amoxicillin+clavulanic
acid has been effective.
122
Pasteurella
P. multocida grow well on blood and chocolate agars but poorly on MacConkey
agar and other media typically selective for gram-negative bacilli. It is susceptible to
a variety of antibiotics. Penicillin is the antibiotic of choice.
Actinobacillus
Legionella
negative bacillus was isolated. Subsequent studies found this organism, named
Legionella pneumophilia, to be cause of multiple epidemic and sporadic infections.
Legionella genus consists 39 species and more than 60 serogroups. L.
pneumophilia is the cause of 85% of all infections; serotypes 1 and 6 are most
commonly isolated.
Members of the genus Legionella are slender, pleomorphic, motile, gram-negative
bacilli. The organisms are nutritionally fastidious; their growth is inhanced with iron
salts and depends on the supplementation of media with L-cysteine. Growth of these
bacteria on supplemented media but not on conventional blood agar media has been
used as the basis for the preliminary identification of clinical isolates. The organisms
are nonfermentative and derive energy from the metabolism of amino acids.
Pathogenesis
Epidemiology
Clinical Diseases
Laboratory Diagnosis
Severe disease treated with azithromycin or levofloxacin; less severe disease can
be treated with erythromycin or tetracycline.
For enviromental sources associated with disease, treat with hyperchlorination,
superheating, or copper-silver ionization.
Eikenella
These organisms colonize the human oropharynx and, in the setting of preexisting
heart disease, can cause subacute bacterial endocarditis.
E. corrodens is a moderate-sized, non-motile, non-spore-forming, facultatively
anaerobic gram-negative bacilli. The organism is fastidious, requires 5% to 10%
carbon dioxide to grow. Small colonies are observed after 48ºC hours of incubation
on blood or chocolate agar. Pitting in agar is a usful differential characteristic, but
fewer than half of all isolates exhibit pitting. The organism also produces a
characteristic bleachlike odor.
E. corrodens is most commonly isolate in the settings of a human bite wound or
fistfight injury and in the periodontal pockets.
Capnocytophaga
Peptostreptococcus
These gram-positive cocci normally colonize the oral cavity, gastrointestinal tract,
genitourinary tract, and skin. They produce infections when they spread from these
sites to normally sterile sites. For example, sinusitis, pleuropulmonary infections,
intra-abdominal infections, endometritis, pelvic abscesses and other infections.
Members of the genus Peptostreptococcus are usually susceptible to penicillin,
metronidazole, imipenem, and chloramphenicol.
Actinomyces
Actinomycosis
Laboratory Diagnosis
Treatment
Propionibacterium
Lactobacillus
Various species of Lactobacillus are part of the commensal flora of human mucous
membranes. These organisms are usually long and sleneder with squared ends, and
frequently occur in chains. They produce quantities of lactic acid during
fermentation, and have been thought to assist in maintaining the acid pH of normal
mucous epithelia. On the other hand, acid production by oral lactobacilli may play a
role in the production of dental caries.
127
CLOSTRIDIUM
TETANUS
The incubation period for tetanus is variable, ranging from 2 to 50 days. The length
of the incubation period is directly related to the distance of the primary wound
infection from the central nervous system. The earliest sign is stiffiness of the jaw
due to spasmic contraction masseter muscles (trismus). Later signs include sustained
contraction of the facial muscles (risus sardonicus) with extension of the spasmic
contractions to the back (opisthotonus), neck, and respiratory muscles. As the disease
progresses, intermittent convulsive seizures are brought on by the slightest
stimulus. Death is due to respiratory complications, usually pneumonia or
asphyxiation.
Diagnosis must be prompt and thus presumptive based upon history of injury and
clinical manifestations. Suspicion should be aroused if stiffiness of the jaw is present.
Primary isolation and identification. If a wound is present, initial anaerobic
cultivation is required on blood agar and in liquid media such as thioglycollate and
cooked meat broth, in which the oxidation-reduction potential is low. Definitive
identification of the organism is based upon the demonstration of a gram-positive rod
with a round and terminal spore that gives the organism a “drumstick” appearance
and by it is production of tetanospasmin as determined by a mouse virulence assay.
The wound must also be cultured aerobically for secondary pyogenic invaders such
as staphylococci and streptococci.
Treatment
These disease are caused by several species of clostridia, but the most common
etiologic agent is C. perfringens, which is also capable of producing food poisoning.
Although there are five types of C. perfringens based upon exotoxin production, the
great majority of human disease is caused by the alpha toxin producing type A.
Clostridial myonecrosis and anaerobic cellulitis are the result of transmission by
means of massive injury with soil contaminated objects or by contaminated surgical
instruments. Food poisoning is the result of transmission by the ingestion of
improperly cooked food contaminated with C. perfringens vegetative cells and left at
room temperature.
The pathogenic potential of C. perfringens is attributed primarily to the 12 toxins
and enzymes. Some of these are listed below:
Lecithinase C (alpha toxin) causing cell membrane disruption and lysis, tissue
necrosis, and edema.
Beta, epsilon, iota, and theta toxins are associated with increased capillary
permeability
Collagenase (kappa toxin) digests and liquefies the collagen in connective tissue.
Hyaluronidase (mu toxin) hydrolyzes the hyaluronic acid of connective tissue
ground substance.
DNase depolymerizes DNA, resulting in tissue liquefaction.
Enterotoxin is a heat-labile protein responsible for diarrheal food poisoning by
inhibiting fluid absorption from the gut.
Laboratory Diagnosis
Treatment
For patients with clostridial myonecrosis and anaerobic cellulitis, prompt and
extensive debridement of the wound is essential. Penicillin or broad spectrum
antibiotics are effective against C. perfringens and secondary pyogenic invaders.
BOTULISM
The two types of botulism, food-borne and infant, are caused by Clostridium
botulinum and occur sporadically.
In food-borne botulism, spores gain access to foods such as vegetables and some
fruits grown in close association with the soil and maintained at a slightly alkaline or
neutral pH during the canning process. The suitable pH, anaerobiasis, and faulty
canning process, provide the favorable conditions necessary for spore germination
and multiplying vegetative cells that produce neurotoxin intracellularly and release it
into the food during cell autolysis. Transmission then occurs when individuals taste
and ingest improperly prepared and improperly cooked canned foods containing pre-
formed neurotoxin.
In infant botulism, infants aged 2 days to 6 months ingest food containing spores
that germinate in the GI tract. Vegetative cells then produce neurotoxin intracellularly
and release it into the gut. The foods responsible for transmission are unknown, but
contaminated honey has been suspected.
Seven antigenically distinct botulinum toxins (A to G) have been described; human
disease is associated with types A, B, E, and F. Only one toxin is produced by most
individual isolates. Like tetanus toxin, C. botulinum toxin have neurotoxic activity. It
is the most powerful exotoxin known, is a protein that is inactivated in food by
boiling for 20 min. The toxin acts by suppressing or blocking the presyneptic release
of acetylcholin responsible for muscle tone.
Clinical Manifestations
period of 8-22 days, ptosis (droopy eyelids), dyspgagia, and fever result. The infants
appears “floppy” due to loss of neck and limb muscle strength.
Laboratory Diagnosis
The rapidity with which death can occur with the food-borne type of botulism
necessitates an immediate diagnosis based upon history (if possible) and clinical
manifestations. For epidemiological purposes, serum specimens and food are
analyzed for neurotoxin by mouse virulence assays. The infant type is usually
diagnosed on clinical grounds, but may be confirmed by demonstrating the
neurotoxin in the stool.
Treatment
MYCOBACTERIUM
These organisms are the most resistant of the nonspore-forming bacteria and are
resistant to drying, remain viable in dried sputum for as long as 8 months, and show a
high degree of resistance to detergents, antibiotics and disinfecting agents. Mycolic
133
acids in the cell wall of the organism contribute to enviromental survival. The
resistance of these organisms to acids and alkalis allow the latter to be used in sputum
concentration techniques employed in the laboratory diagnosis of tuberculosis.
The basic structure of the cell wall is typical of gram-positive bacteria: an inner
cytoplasmic membrane overlaid with a thick peptidoglycan layer and no outer
membrane. The peptidoglycan sekeleton is covalently linked with arabinogalactan.
This layer is overlaid with polypeptides and a hydrophobic layer of highly antigenic
mycolic acids consisting of free lipids, glycolipids, and peptidoglycolipids. These
lipids constitute 60% of the dry weight of the cell wall. The peptide chains in the
outer layer constitute 15% of the cell weight and are biologically important antigens,
stimulating the patient’s cellular immune response to infection (Fig. 35).
Mycobacterium are strict aerobes tha grow best on potato-egg (e.g., Lowenstein-
Jensen) and serum agar base (e.g., Middlebrook 7H10) media, both of which contain
malachite green dye to inhibit the growth of other bacteria. Optimal growth occurs on
or in these media at 37ºC in the presence of 5-10 carbon dioxide. The organisms are
relatively slow growers. On solid media, colonies appear as dry, wrinkled, and cream
colored (nonpigmented) after 10 days to 8 weeks. The production of niacin by
M.tuberculosis is an important biochemical reaction in the laboratory identification of
the organism, inasmuch as nonpathogenic and other pathogenic or potentially
pathogenic mycobacteria do not produce niacin.
Mycobacterium tuberculosis
terminal airways. At these sites, the bacteria penetrate into unactivated alveolar
macrophages. The phagocytized bacilli inhibit acidification of the phagosome and
subsequent phagosome-lysosome fusion, and proceed to replicate freely (it is unclear
whether the bacilli replicate in the phagosome or in the cytoplasm). The infected
phagocytic cells are eventually destroyed, after which there are further cycles of
phagocytosis by macrophages, mycobacterial replication, and cell lysis.
Although phagocytosis is initiated by alveolar macrophages, circulating
macrophages and lymphocytes are attracted to the infectious focus by the bacilli,
cellular debris, and host chemotactic factors (e.g., complement component C5a). The
histologic characteristic of this focus is formation of multinucleated giant cells of
fused macrophages, also called Langhans’ cells. Infected macrophages can also
spread during the initial phase of disease to the local lymph nodes as well as into
blood stream and other tissues (e.g., bone marrow, spleen, kidneys, central nervous
system).
Three to four weeks after exposure to the organisms, DTH and CMI develop. The
great majority of individuals develop a relatively high degree of CMI by a
mechanism in which alveolar macrophages, activated by the lymphokine MAF
(macrophage activating factor) released from T lymphocytes, phagocytize and digest
the organisms. An adequate CMI response results in healing of most infected sites by
fibrosis and calcification, although calcified areas may still harbor tubercle bacilli for
years or for life. The calcified primary site lesions, including those in lymph nodes,
are visible on X-ray and referred to as the Ghon complex.
Epidemiology
pulmonary disease, (2) positive skin test reactivity, and (3) the laboratory detection of
mycobacteria either with microscopy or in cultures.
Extrapulmonary tuberculosis resulting from hematogenous spread of the bacilli
during the initial phase of multiplication can also be seen. The most common sites of
infection include lymph nodes, pleura, and the genitourinary tract. With disseminated
or miliary tuberculosis there is frequently no evidence of pulmonary disease.
Laboratory Diagnosis
Treatment
Mycobacterium leprae
Clinical Manifestations
Following introduction of M. leprae into the host, the organisms multiply slowly
inducing a chronic granulomatous response characterized by an influx of
mononuclear cells. After an incubation period of 3-5 years or longer, the disease
manifests as the lepromatous or tuberculoid type with three intermediate stages.
In lepromatous leprosy, the lesions occur as large maculae in cooler body tissues
such as skin (especially nose and outer ears), testicles, and superficial nerve endings.
The course of lepromatous leprosy is slow but progressive. Many macules, papules
or nodules; extensive tissue destruction (e.g., nasal cartilage, bones, ears); lack of
nerve enlargement occurs.
Epidemiology
Laboratory Diagnosis: History and clinical manifestations are useful adjuncts but
not definitive in the diagnosis of leprosy.
Specimens depend on the disease process and include skin lesion biopsies
(including the ear lobes) and nasal secretions. Definitive identification is based upon
the demonstration of typical acid-fast bacilli within phagocytic foam cells; numerous
packets of organisms or “globi” are observed in lepromatous leprosy, but are difficult
or impossible to observe in the tuberculoid type.
138
Treatment
Although at one time the sulfone drug dapsone was the antimicrobial of choice in
the treatment of leprosy, the development of a high degree resistance has prompted
the recommendation of combined dapsone, rifampin, and clofazimine for patients
with lepromatous leprosy and dapsone and rifampin for thoes with the tuberculoid
type.
NOCARDIA
SYSTEMIC NOCARDIOSIS
The most important gram-negative anaerobes that colonize the human oral cavity
(particularly the gingival sulcus), female genital tract, and gastrointestinal tract are
the bacilli in the gener Bacteroides, Fusobacterium, Porphyromonas, and Prevotella.
Anaerobes are the predominant bacteria at each of these sites, outnumbering aerobic
bacteria by 10-to 1000-fold. Despite the abundance and diversity of these bacteria,
most infections are caused by relatively a few species. Among these pathogens, the
most important is Bacteroides fragilis, the prototypical endogenous anaerobic
pathogen. Because these organisms are unable to survive in the presence of
atmospheric oxygen, they cannot be passed easily from patient to patient. Thus, these
organisms cause disease almost exclusively in their original hosts.
A few species of anaerobes, notably some spore-forming species, clostridia, are
aerotolerant. The anaerobic gram-negative bacilli group, are not aerotolerant but are
139
able to survive some period of oxygen exposure in the original specimen and on an
agar plate once colonies have matured.
In colonial morphology a few species of this group bacteria have very distinctive
colonies that aid in their identification on the culture.
The B. fragilis group yield large, shiny, opaque colonies on blood agar.
Most species of Porphyromonas and some species of Prevotella, a gram negative
bacilli, are pigmented. They use the heme component of RBCs in the medium to
generate porphyrin compounds, which become components of various dark pigments.
Colonies containing porphyrins will fluoresce under UV light before the pigments
have developed.
Colonies of some strains of F. nucleatum show typical bread-crumb appearance.
colonies are shiny white and opaque.
Anaerobes are identified by morphology, enzymatic and biochemical tests, the
production of specific end products of glucose metabolism as detected using gas-
liquid chromatography (GLC) of spent culture medium, and by cell wall fatty acid
composition. Definitive identification of many species of anaerobes can only be
performed in specialized laboratories. However, rapid 2-day presumptive
identification is within the capabilities of any clinical microbiology laboratory.
Pathogenesis
Each of the anaerobic species have adherence factors that allow them to multiply
on mucous membrane surfaces and many of them produce proteolytic and lipolytic
enzymes.
Lipopolysaccharide. The gram-negative anaerobes contain LPS in their cell walls.
The LPS of fusobacteria appears similar to that of aerobic gram-negative bacteria, but
the LPS of members of the B. fragilis group is distinctly different. Both
Fusobacterium and Bacteroides LPS contributes to abscess formation.
The polysaccharide capsule of B. fragilis has been extensively studied as the
principal virulence factor of these strains. The capsule is antiphagocytic and promotes
abscess formation. Other genera, such as Fusobacterium and Prevotella, also include
encapsulated species.
Fimbriae. Several species of anaerobic gram-negative bacilli have been shown to
posess pili or fimbriae, which mediate attachment to epithelial cells.
Hemagglutinin. B. fragilis possesses a surface hemagglutinin, which may aid in
invasion of host tissues.
Extracellular products associated with pathogenicity are:
Laboratory Diagnosis
differential plate for rapid identification of the B.fragilis group; and a broth
enrichment backup culture.
3. All cultures are incubated anaerobically for 2-14 days before they are examined.
4. Isolates are identified based on colony morphology, Gram stain morphology,
susceptibility to antibiotics, constitutive enzymes, biochemical reactions, and by the
end products of glucose metabolism. A few rapid tests can identify most isolates to
genus within 2 days of observing isolated colonies.
Gas-liquid chromatographic analysis of volatile and nonvolatile fatty acids
produced by metabolic processes of anaerobes is the definitive identification method.
The cumbersome nature of the methods and the expense of the apparatus precludes
the use of thses tests in most clinical laboratories.
Treatment
SPIROCHETES
TREPONEMA, BORRELIA, AND LEPTOSPIRA
TREPONEMA
The treponemal species responsible for human disease are Treponema pallidum
and T. carateum.
T. pallidum is subdivided into subspecies pallidum, the etiologic agent of syphilis,
subspecies pertenue, the agent responsible for yaws, and subspecies endemicum, the
etiologic agent of endemic syphilis.
Although DNA homology between Treponema pallidum subsp. pallidum and
Treponema pallidum subsp. pertenue is almost 100%, subspecies nomenclature is
based on differences in their clinical manifestations and geographic locations. The
variant status of Treponema pallidum subsp. endemicum has warranted subspecies
classification for this organism.
T. pallidum subsp. pallidum is an obligate parasite of humans and does not occur in
nature or in animals. The organism is a thin, coiled spirochete (0.1 x 5-15 µm)
actively motile, exhibiting a shimmering motion in serious exudate and a directional,
corkscrewlike motion in fibrinous exudate. The spirochetes cannot be seen with light
microscopy in specimens stained with Gram or Giemsa stains, but can be best
recognized and studied by dark field microscopy.
None of pathogenic treponemes has been cultured in/on artificial media. Limited
growth has been achieved in cultured rabbit epithelial cells but replication is slow and
can be maitained only for a few generations.
Treponema pallidum is extremely labile, unable to survive exposure to drying or
disinfectants. Thus inanimate objects such as toilet seats cannot contribute to the
spread of syphilis. Direct person-to-person contacts is required for transmission.
Syphilis. This occurs worldwide and is the third most common sexually
transmitted disease in the developed world. The organism enters the body via skin or
mucous membrane abrasions. Localized multiplication occurs, resulting in
inflammatory cell infiltration, followed by endarteritis. The infection is divided into
three phases:
1. Primary Stage. This stage of the disease occurs after an incubation period
ranging from 10 to 90 days, but usually 3 weeks. Primary syphilis is
characterized by the appearance of painless, indurated, well-circumscribed ulcer,
known as chancre, accompained by a regional lymphadenopathy that is usually
bilateral. At this time, the chancre and lymph nodes contain treponemes and the
disease is communicable. The primary lesion heal in 1-6 weeks, presumably due
to CMI. During this local healing process, organisms within the deeper tissues
144
Laboratory Diagnosis
2. Specific antibody tests: These tests are based on T. pallidum antigens and are
used to confirm non-specific screening tests. Commonly used tests include the
fluorescent treponemal antibody (FTA) test and the T. pallidum
microhemagglutination assay (TP-MHA), in which erythrocytes coated with T.
pallidum antigen are agglutinated by serum from patients with antibodies to T.
pallidum.
Specific tests to detect IgM antibodies are used to diagnosed congenital
infections.
Treatment
Penicillin is the drug of choice in the treatment of all stages of syphilis and
congenital syphilis. Penicillin resistant strains of T. pallidum have not been reported.
Tetracycline, erythromycin, or chloramphenicol is administered if the patient is
allergic to penicillin.
Treponema carateum
BORRELIA
Borrelia are associated with two important human infections, relapsing fever
(primarily Borrelia recurrentis) and Lyme disease (Borrelia burgdorferi).
Members of the genus Borrelia are identical appearing as loosely coiled, thin,
flexible, actively motile spirochetes 3-30 µm in length and 0.2-0.5 µm in diameter.
These organisms are gram-negative and stain well with Giemsa and Wright stains,
and can be easily seen in smears of peripheral blood collected from patients with
relapsing fever.
Borrelia are microaerophilic and have complex nutritional requirements, making
recovery in culture difficult. The few species that have been successfully cultured
146
Members of the genus Borrelia are responsible for two important human diseases:
relapsing fever and Lyme disease.
develops at the site of tick bite. Chronic neurologic, cardiac, and rheumatic
manifestations are also occurs after skin lesion.
Laboratory Diagnosis
Microscopy. Because of their relatively large size, the borreliae responsible for
relapsing fever can be seen during the febrile period by preparing a Giemsa stain of
the blood. In contrast, the microscopic examination of blood or tissues collected from
patients with Lyme disease connot be recommended becaus B. burgdorferi is rarely
seen in clinical specimens.
Culture. The cultures are rarely performed in most clinical laboratoreis because
the media are not readily available and replication is slow. The in vitro isolation of B.
burgdorferi has had limited success.
Serology. Because the borreliae responsible for relapsing fever undergo antigenic
phase variation, serlogic tests are not useful. In contrast, serologic testing is an
important confirmatory test for patients who clinically diagnosed as have Lyme
disease.
Treatment
LEPTOSPIRA
Laboratory Diagnosis
Treatment
Penicillin and tetracycline are effective if administered within the first 2 – 4 days
after initial onset of clinical manifestations. Later administration has no effect upon
the course of the disease.
RICKETTSIACEAE FAMILY
The family Rickettsiaceae consist of aerobic, gram-negative small (0.3 x 1-2 µm)
bacilli that, with one exception (Rochalimaea), are obligate intracellular parasites;
they grow only in yolk sac of embryonated eggs, tissue cultures and in animal
models.
Four genera (Rickettsia, Coxiella, Rochalimaea, and Ehrlichia) are associated with
human disease. The pathogenic species are maintained in animal reservoirs and
transmitted by arthropod vectors (ticks, mites, lice, fleas). Humans are usually
accidental hosts.
the Gram stain. With the exception of Rochalimaea, all rickettsia are strict
intracellular parasites. The intracellular location of the rickettsia vary: Rickettsia
species are generally found free in the cytoplasim, whereas Coxiella and Ehrlichia
multiply in cytoplasmic vacuoles. Rickettsia rickettsii, the bacterium responsible for
Rocky Mountain spotted fever, can also grown to high concentrations in the nucleus.
Rochalimaea multiplies readly on the surface of eukaryotic cells but rapidly dies
following phagocytosis.
The rickettsia enter eukaryotic cells by phagocytosis. After phagocytosis occurs,
rickettsia must be released into the cytoplasm or the organism fails to survive. A
multiplication by binary fission then proceeds slowly compared with other bacteria
(generation time, 6 – 10 hours) untile destruction of the host cell ocuurs, freeing the
rickettsia to infect new cells. In contrast with the other rickettsia, multiplication of
Coxiella and Ehrlichia proceeds within phagolysosomes rather than in the cytoplasm,
and host cell lysis is low.
Once released from the host cell, most rickettsia are unstable and die quickly. The
exception is Coxiella, which is highly resistant to desiccation and remains viable in
the enviroment for months to years. This characteristic is extremely important in the
epidemiology of Coxiella infections.
Rickettsial Infections
Q Fever usually presents as an acute febrile illness with headache and frequently
hepatitis. Although tick-borne Q fever is known, most cases are acquired by aerosols.
The etiologic agent, Coxiella burnetii, is shed in the feces and genital secretions of
infected domestic animals, such as sheep, cattle, and goats. Because of it is resistance
to harsh enviromental conditions, the organism remains viable in aerosols and causes
disease when inhaled by humans.
Ehrlichiosis. Canine ehrlichiosis is transmitted by the dog tick and is
characterized by an acute phase of pancytopenia and a chronic phase with fever,
anemia, and occasional bleeding from the capillaries.
Laboratory Diagnosis
CHLAMYDIACEAE FAMILY
Members of the family Chlamydiaceae are obligate intracellular bacteria that were
once regarded as viruses. Chlamydia possess inner and outer membranes similar to
those of gram-negative bacteria, contain both DNA and RNA, possess prokaryotic
ribosomes, synthesize their own proteins, nucleic acids, and lipids, and are
susceptible to numerous antibiotics. Unlike other gram-negative bacteria, however,
chlamydiae lack a peptidoglycan layer between the inner and outer membranes and
undergo a unique growth cycle.
The genus Chlamydia is divided into three species: C. trachomatis, C. pneumoniae
and C. psittaci. Classification is based on the basis of antigens, the susceptibility to
sulfonamides, the morphology of the elementary bodies, the formation of inclusions
containing glycogen, and the natural host range.
151
Life cycle:
· Ebs bind to specific host cell receptors and enter by endocytosis. Target cells include
conjunctival, urethral, rectal and endocervical epithelial cells.
· Intracellular Ebs remain within phagosomes and replicate. Lysosomal fusion with
the phagosome-containing Ebs is inhibited, probably by chlamydial cell wall
components.
· After approximately 8 h, Ebs become metabolically active and form RBs, which
synthesize DNA, RNA and protein utilizing host cell adenosine triphosphate (ATP) as
energy source.
· RBs undergo multiple division and the phagosome becomes an “inclusion body”
(visible by light microscope), which is contain as may as 1000 RBs. Glycogen-
containing inclusion bodies, such as those formed only by C.trachomatis, stain
orange-brown with iodine.
· After approximately 24 h, the RBs recognize into smaller Ebs and, after a further 24
– 48 h, the host cell lyses and infective Ebs are released.
C.trachomatis
Infections of C.trachomatis
cause constant abrasion of the cornea, pannus formation (invasion of vessels into the
cornea), and loss of vision. Trachoma is transmitted eye-to-eye by droplet, hands,
contaminated clothing, and by eye-seeking flies, which transmit ocular discharges to
eyes of other person. Infections occurs predominantly in children.
Adult inclusion conjunctivitis. An acute follicular conjunctivitis caused by
serotypes A, B, Ba, D to K associated with genital infections. The infection is
characterized by mucopurulent discharge, keratitis, corneal infiltrates, and
occationally some corneal vascularization. Most cases occur in adults between the
ages of 18 and 30, with probable genital infection before eye involvement. Infected
secretions from the genital tract spread the infection through sexual activity,
including anal-genital sex, and by direct contact. Adults can transfer the organism to
their eyes after touching their genital tracts. Infants usually acquire the disease during
passage through the infected birth canal of the mother.
Neonatal conjunctivitis. Inclusion conjunctivitis in the newborn is acquired by
passage through an infected maternal birth canal and associated with serotypes D—
K. After an incubation period of 5—12 days, swelling of the lids, hyperemia, and
copious purulent discharge appear. Without therapy or with tropical therapy only,
infants are at risk for development of C. trachomatis pneumunia.
Urogenital infections are associated with serotypes D—K. The most common
cause of sexually transmitted disease in the developed world.
Most genital tract infections in women are asymptomatic but can nevertheless
spread to cause symptomatic disease, including cervicitis, endometritis, urethritis,
salpingitis, and perihepatitis. Symptomatic infections produce mucopurulent
discharge and hypertrophic ectopy and generally yield greater numbers of organisms
on culture than do asymptomatic infections.
Unlike genital infections in women, the majority of genital infections in men
caused by C. trachomatis are symptomatic. Approximately 35% to 50% of cases of
nongonococcal urethritis are caused by C. trachomatis. Postgonococcal urethritis
results from coinfection with both N. gonorrhoeae and C. trachomatis, and
symptomatic illness develops after successful treatment of the gonorrhea because of
the longer incubation period of chlamydiae. Epididymitis may occur in association
with urethritis due to chlamydiae or coliform bacteria.
Lymphogranuloma venereum (LGV) is a chronic sexually transmitted disease
caused by C. trachomatis serotypes L1, L2, and L3. The disease is highly prevalent in
tropical climates (Africa, Asia, and South America).
After an incubation period of 1—4 weeks, a prima lesion (ulcer) may appear at the
site of infection (e.g., penis, urethra, glans, scrotum, vaginal wall, cervix, vulva).
The second stage of infection is marked by inflammation and swelling of the
lymph nodes draining the site of initial infection. Most commonly, the inguinal nodes
are involved, producing painful, fluctuant “buboes” that gradually enlarge.
153
Laboratory Diagnosis
Culture: Swabs from the affected site collected in a special transport medium are
inoculated into tissue culture. Growth of C. trachomatis is recognized by Giemsa
staining to show intracellular bodies or by immunofluorescence staining with specific
antisera.
Direct antigen detection can be made by immunofluorescence or enzyme-linked
immunosorbent assay (ELISA) methods. Both techniques utilize labelled specific
antibodies to C. trachomatis.
Serology: Antibodies to C. trachomatis may be detected by complement fixation or
micro-immunofluorescence tests.
· Assays based on the polymerase chain reaction and ligase chain reaction are also
now available.
Treatment: Since asymptomatic genital infection is common, all partners of
infected patients should be treated. Tetracycline or doxycycline, followed by
erythromycin and sulfasoxazole are still the therapies of choice.
C. psittaci
C. pneumoniae
Associated Infections
· M. pneumoniae: pharyngitis; primary atypical pneumonia
· Other mycoplasmas, including M. hominis and U. urealyticum, are found in human
genital tracts. These organisms cause postpartum fever and may contribute to
infertility. M. hominis can cause postsurgical wound infections.
Laboratory Diagnosis
VIROLOGY
Virus Structure
The electron microscope shows that viruses censist of a protein coat (capsid) and a
core of nucleic acid. The larger viruses have an envelope composed of lipid,
proteins, and glycoproteins (Fig. 35). This lipid envelope can be disrupted by
detergents and solvents such as ether, which will then inactivate the virus. Some
viruses also contain enzymes required for replication.
Capsid
The capsid is necessary for morphogenesis of the virus. It is also determine host
specificity, protects the viral genome, enhances the efficiency of infection, and
induces the formation of neutralizing antibody.
Genome
All true viruses have a nucleic acid that is either DNA or RNA. The genome may
consist of double-stranded or single-stranded DNA or RNA and contains all necessary
information for virus replication in susceptible cells.
Envelope
Lipid bilayer membrane surrounding the nucleocapsid of some viruses (enveloped
viruses). This viral envelope is derived from the membrane of the host cell except
that membrane proteins of the host are largely replaced by viral proteins such as:
1. Glycoproteins (peplomers) extend from the surface of the virion and for many
viruses can be observed as spikes. The peplomers play a key role in viral
attachment to a susceptible cell and in the induction of neutralizing antibodies
against the enveloped virus.
2. Matrix proteins found between the nucleocapsid and envelope. These proteins
strength the virion and facilitate their assembly.
Virion
A virus particle with all it is morphologic components is known as a virion
VIRUS CULTIVATION
Viruses are obligate, intracellular parasites and thus can only replicate in living
cells. Viruses utilize the host cell metabolism to assist in the synthesis of viral
proteins and progeny virions; the host cell range of viruses may be narrow or wide.
For diagnostic purposes, most viruses are grown in cell cultures, either secondary
or continuous cell lines; the use of embryonic eggs and laboratory animals for virus
culture is reserved for specialized investigations. Cell culture lines are often derived
from monkey kidney, human fetal lung, human amnion, and human cancer cells.
Virus replication in cell cultures may be detected by :
1. Cytopathic effect (CPE): some viruses can be recognized by their effect on cell
architecture, e.g. changes in cell morphology, cell lysis, the presence of inclusion
bodies, or the formation of multinucleated cells (syncytia formation). Inclusion
bodies are histologic changes in cells caused by the presence of viral components or
changes in cell structure.
2. Haemadsorption: viruse expressing haemagglutinins on the cell surface may be
recognized by absorption of red blood cells to infected cells.
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Viras replication in host cells involves the following steps (Fig. 36).
1. Attachment (adsorption) occurs of the viral nucleocapsid (naked viruses) or of
virus envelope components (enveloped viruses) to cell has specific viral receptors.
2. After attachment, the nucleocapsid enters the cell by endocytosis or by the fusion
of the cell membrane with viral envelope. This fusion is usually mediated by a viral
envelope protein.
3. Uncoating follows, which involves the proteolytic removal of viral protein coat
and liberation of nucleic acid. The disassembly is achieved by unidentified host
enzymes.
4. Synthesis of viral proteins. In order to direct the host cell ribosome to produce
viral proteins (genome, capsid), virus specific mRNA must be produced. The
mechanisms for virus specific mRNA production depend on the viral genome type.
a) RNA or DNA
b) Single-stranded or double-stranded
c) Positive sense (base sequence configured as required for translation = mRNA)
or negative sense (base sequence requires transcription).
5. Morphogenesis and maturation occur with assembly of components (nucleic
acid, proteins) to form viral particles.
6. Release of the virus is by bursting of infected cells (lysis) or by budding through
plasma membrane (host cell does not necessarily lose viability, therefore it can shed
viral particles for extended periods).
With some viruses, e.g. hepatitis B, the host cell remains viable and continues to
release virus particles or subviral antigens at a slow rate. These persistent infections
acts as a continuing source of new infectious viruses.
During latent infections the virus does not undergo replication; the viral nucleic
acid may remain in host-cell cytoplasm (e.g. herpes virus) or become incorporated
into the host genome (e.g. human immunodeficiency virus HIV). A trigger is required
to recommence viral nucleic acid replication, transcription and translation.
The key viral product in the synthetic phase is the viral mRNA. The pathways
leading from viral genomes to multiple copies of viral mRNA can be best be
described under the following three categories:
1. RNA Viruses Other Than Retroviruses. It is important to remember that the host
cell does not have an RNA dependent RNA polymerase; therefore, this polymerase
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must be encoded in the viral genome. The initial step in the synthetic phase depends
on whether the viral genome is single or double stranded; and, if single stranded,
whether it is positive or negative sense.
Viruses with single-stranded positive sense RNA (e.g., Picornaviridae) acts as
mRNA, bind to ribpsomes, and direct protein synthesis. Then RNA dependent RNA
polymerase is produced. Using the viral genome as template, this polymerase
synthesizes a complimentary strand of RNA. Together, the original viral genome
(positive sense) and the newly synthesized complimentary strand (ngative sense)
form a double-stranded RNA known as the replicative form. Using this replicative
form as the template, the viral polymerase synthesizes from the negative sense strand
many copies of positive sense RNA. These positive sense RNAs serve as viral mRNA
as well as viral genomes for progeny virions.
Viruses with single-stranded negative sense RNA (e.g., Orthomyxoviridae) cannot
be translated. It must be transcribed into positive sense RNA. This initial step is
performed by an RNA dependent RNA polymerase found in the capsid. Once positive
sense RNA are formed, the synthetic phase proceeds as described for Picornaviridae.
Viruses with double-stranded RNA (e.g., Reoviridae). The capsid also contains an
RNA dependent RNA polymerase that transcribes the negative sense of the uncoated
viral genome into positive sense RNA
VIRUS GENETICS
Genetic interaction. When a single cell is infected by two strains of viruses of the
same species, genetic recombination may occur. Both strains of virus contribute a
part of their genomes to the recombinant.
2. Interference. Both viruses compete for limiting amount of viral or cell protein
essential for virus replication. Consequently, the formation of progeny virions from
one or both parentral viruses is partially or completely inhibited.
3. Phenotyping Mixing. The genome of a virus may be wrapped in proteins supplied
partly or completed by the other virus.
VIRAL DISEASE
Viral disease in the body progresses through defined steps, just like viral
replication in the cell.
The early steps are as follows:
1. Acquisition (entry into the body)
2. Initiation of infection at a primary site.
3. An incubation period, when the virus is amplified and may spread to a secondary
site
The incubation period may proceed without symptoms (asymptomatic) or may
produce nonspecific early symptoms, teremed the prodrome. The symptoms of the
disease are caused by tissue damage and systemic effects caused by the virus and
possibly the immune system. These symptoms may continue through the
convalescence, while the body repairs the damage.
The virus gains entry into the body through breaks in the skin or through the
mucoepithelial membranes that line the orifices of the body (eyes, respiratory tract,
mouth, genitalia, and gastrointestinal tract). The skin is an otherwise excellent barrier
to infection, and the orifices are protected by tears, mucus, ciliated epithelium,
stomach acid, bile, and immunoglobulin A. Inhalation is probably the most
common route of viral Infection
On entry into the body, the virus replicates in cells that express viral receptors and
that have the appropriate biosynthetic machinery. Many viruses initiate infection in
the oral mucosa or upper respiratory tract. Symptoms may accompany viral
replication at the primary site. The virus may replicate and remain at the primary site,
may disseminate to other tissues via the blood stream or the mononuclear phagocyte
and lymphatic system, or may disseminate through neurons.
The blood stream and the lymphatic system are the predominant means of viral
transfer in the body. The transfer of virus in the blood is termed viremia. The virus
may be free in the plasma or may be cell-associated in lymphocytes or macrophages.
Viruses teken up by phagocytic macrophages may be inactivated, may replicate, or
may be delivered to other tissues by way of the mononuclear phagocyte system.
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Viruses can gain access to the central nervous system or brain (1) from the blood
stream, (2) from infected meninges or cerebrospinal fluid, or (3) by means of the
migration of infected macrophages or the infection of peripheral and sensory neurons.
The skin is best barrier to infection, but openings in the skin, whether natural
orifies (e.g., mouth, eyes, nose, ears, anus) or due to trauma such as abrasion or
puncture, provide pathogens with access to the body. After the virus penetrates the
natural barriers, it activates the antigen-nonspecific (innate) immune defenses (e.g.,
fever, interferon, macrophages, natural killer cells), which attempt to limit and
control local viral replication and spread. Antigen-specific immune responses (e.g.,
antibodies, helper T cells) are the last to be activated. Interferon and cytotoxic T-cell
responses may have involved primarily as antiviral defence mechanisms.
The ultimate goal of the host response is to eliminate the virus and the cells
harboring or replicating the virus. The immune response is the best and in most
cases the only means of controlling a viral infection. Both humoral and cellular
immune response are important for antiviral immunity.
ANTIVIRALS
Antivirals are substances capable of intercepting the virus replication cycle. There
are literally hundreds of antivirals; but only a few have been approved by the FDA for
the prophylaxis or treatment of specific virus infections in humans. The antivirals are
grouped according to their modes of antiviral actions.
VIRUS CLASSIFICATION
1. Direct (detection of virus particles, viral antigen, viral lesions or nucleic acid) by:
microscopy; electron microscopy; particle agglutination; immunofluorescence;
serology; PCR and RT-PCR.
2. Indirect (detection of virus-specific host response) by serology (complement
fixation test, haemagglutination inhibition test ELISA etc).
DNA VIRUSES
The herpesvirus family is one of the most important family of viruses in medical
practice. These viruses are ubiquitous, infect virtually every person, induce a wide
variety of diseases, respond to antiviral therapy, and enter into latency after recovery
from primary infections. There are eight species of human pathogens in this family of
viruses: herpes simplex 1 (HSV 1), herpes simplex 2 (HSV 2), varicella-zoster
(VZV), Epstein-Barr (EBV), cytomegalo virus (CMV), human herpes 6 and 7 (HHV
6 and HHV 7), and the recently discovered human herpes 8 (HHV 8) associated with
Kaposi’s sarcoma.
Primary targets of HSV are the skin and mucosa. The virus multiplies at the
implantation site, damages the tissue, and forms a vesiculopapule. Lesions are usually
not painful unless irritated. On moist areas, herpetic lesions usually ulcerate forming
shallow ulcers. HSV is transmitted by direct contact via vesicle fluid, saliva, and
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gingival secretions. Common sites for herpetic lesions are the skin around the oral
and genital orifices, the oral cavity, the cornea, the vagina and cervix, the anal canal,
and the urethra.
Oral herpes can be caused by HSV-1 or HSV-2. Primary herpetic
gingivostomatitis in toddlers and children is almost always caused by HSV-1,
whereas young adults may be infected with HSV-1 or HSV-2. The lesions begin as
clear vesicles that may be widely distributed throughout the mouth, involving the
palate, pharynx, gingivae, buccal mucosa, and tongue.
People may experience recurrent mucocutaneous HSV infection (cold sores, fever
blisters) even though they never had clinically apparent primary infection. The
lesions usually occur at the corners of the mouth or next to the lips. Symptoms of the
recurrent episode are less severe, more localized, and of shorter duration than those of
a primary episode.
Herpetic keratitis is almost always limited to one eye. It can cause recurrent
disease, leading to permanent scarring, corneal damage, and blindness.
Herpetic whitlow is an infection of the finger. The virus establishes infection
through cuts or abrasions in the skin. Herpetic whitlow often occurs in nurses or
physicians who attend patients with HSV infections.
Eczema herpeticum is acquired by children with active eczema.
Genital herpes is usually caused by HSV-2 but can also be caused by HSV-1
(responsible for 10 % of genital infections). Most primary genital infections are
asymptomatic.
Herpes encephalitis is an acute febrile disease that is usually caused by HSV-1.
This disease occurs at all ages and at any time of the year.
HSV meningitis is most often a complication of genital HSV-2 infection, and
symptoms resolve on their own.
HSV infection in the neonate is a devastating and usually fatal disease caused
most orten by HSV-2. It may be acquired in utero but more commonly is contracted
either during passage of the infant through the genital canal or postnatally from
family members or hospital personnel.
Treatment: The drug of choice is acyclovir. It is a potent inhibitor against HSV 1
and HSV 2 and is virtually nontoxic to humans. No vaccine or preventable drug is
available.
Most transmission of EBV occurs by intimate contact with saliva that contains
virus. EBV replicates in mucosal epithelium. The virus then spreads to the lymph
nodes, where it infects B cells. EBV next travels via the blood to other organs,
particularly targeting liver and spleen. The B cell infection is an abortive one, leading
to B cell proliferation, accompanied by nonspecific increases in total IgM, IgG, and
IgA.
Infectious mononucleosis (IM). The “atypical lymphocytosis” characteristic of
IM is caused by the active cytotoxic T cell response to the EBV antigens expressed
by infected B cells. The typical IM syndrom appears after an incubation period of
four to seven weeks, and includes fever, malaise, pharyngitis, lymphadenopathy
(swollen glands), and, often, hepatosplenomegaly.
Treatment is by acyclovir. No vaccine or preventable drug is available.
HHV-6 was first isolate from the blood of patients with AIDS and grown in T-cell
cultures. In 1988, HHV-6 was serologically associated with a common disease of
children, exanthema subitum, commonly known as roseola.
HHV-7 was isolated in a similar manner from the T-cells of a patient with AIDS
who was also infected with HHV-6. However, HHV-7 remains an orphan virus with
no disease association
Human Herpesvirus 8
Poxviruses are a large, complex group of viruses that cause disease in humans and
other animals. Many of the basic characteristics of the poxviruses are uniquely
different from those of other viruses. Some virologists have questiond the validity of
classifying the poxviruses as viruses.
Poxvirus are the largest viruses, measuring 230-300 nm in size, and are ovoid to
brick-shaped. They have a capsid that is referred to as complex because it hase
neither helical nor icosahedral symmetry. An outer membrane and envelope enclose
the core and core membrane, which are flanked by two lateral bodies of unknown
function. The viral genome is a double-stranded DNA.
Three species are of medical interest: smallpox, vaccinia, and molluscum
contagiosum viruses. Smallpox has been eradicated through a worldwide effort of
vaccination case finding and quarantine. Vacciniavirus is the smallpox vaccin virus.
Smallpox vaccination is not an innocuous procedure and is no longer recomended for
universal usage. The vaccinvirus is now used extensively as vectors for selected
genes (e.g., HbsAg, HIV gp 120, ect.). Molluscum contagiosum virus causes a benign
self-limiting skin lesion characterized by umbilicated pearlike nodules.
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Disease of Papovaviruses
Warts
A warts is a benign, self-limited proliferation of skin that progresses with time. The
appearance of the wart (domeshaped, flat, or plantar) depends on the HPV type and
the infected site.
Warts spontaneously regress, but the regression may take many months to years.
Warts are removed because of pain and discomfort, for cosmetic reasons, and to
prevent to spread to other parts of the body or to other people.
At present, the best way to prevent transmission of warts is to avoid coming in
direct contact with infected tissue.
RNA VIRUSES
Orthomyxoviruses are the etiologic agent of influenza, which is one of the last few
major epidemic diseases that inflict human.
Types and subtypes of the family. Influenza viruses are classified as types A, B,
and C depending on their inner proteins, mainly the M proteins. Thus, all type A
viruses share common internal antigens that are distinct from those shared by all type
B viruses. Only the type A viruses are broken down into subtypes. The classification
into subtypes depends on antigens associated with the outer viral proteins, H and N.
Taking into consideration animal as well as human influenza viruses, 14 H and 9 N
subtypes have been described. However, among human influenza viruses, only three
H (H1, H2, and H3) and two N (N1 and N2) subtypes are found. Human influenza
viruses are therefore designated, for example, as subtype H1N1, H2N2, H3N2, etc.
1. Major antigenic change (antigenic shift) is due to the replasement of one H (or
N) gene by another. After the shift, H of the virion is totally different
immunologically from those before the shift. This process occurs only with the
influenza A. Such changes are often associated with the occurence of pandemics.
Antigenic shifts occur infrequently, taking place on average 10 years.
2. Minor antigenic change (antigenic drift) is due to mutation of the H (or N) gene
of types A and B viruses. This process occurs every 2 to 3 years, causing local
outbreaks of influenza A and B infection. Immunity acquired through infection with
parentral virus is less effective against the mutant virus.
Epidemiology
Influenza Type A.
The primary target of the influenza virus type A is the respiratory tract. The virus is
deposited on the respiratory tract through inhalation of droplets that contain the virus
or through the direct contact with respiratory secretion positive for the virus.
After an incubation period of 1 to 4 days, the “flu syndrome” begins with a brief
prodrome of malaise and headache lasting a few hours. The prodrome is followed by
the additional abrupt onset of fever, severe myalgia, and usually a nonproductive
cough. The illness persists for approximately 3 days, and unless a complication
occurs, recovery is complete within 7 to 10 days.
Influenza in young children resembles other severe respiratory tract infections,
causing bronchiolitis, croup, otitis media, and rarely febrile convulsions.
Complications of influenza include bacterial pneumonia and myositis
Early diagnosis and prompt treatment of secondary bacterial pneumonia are
important in the management of influenza. Annual administration of influenza vaccin
(inactivated) provides about 80% protection. Amantadine, if given within 1 or 2 days
after onset, is effective in shortening the course and reducing the severity.
Influenza Type C. Type C virus infections are uncommon and have little medical
importance.
Laboratory Diagnosis
Paramyxoviruses are enveloped, have helical nucleocapsid, and vary from 100 to
300 nm in diameter. The genomes of the virus family are single-stranded, negative
sense, and nonsegmented RNA.
This family of viruses includes the following human pathogens: measles, mumps,
respiratory syncytial, and parainfluenza viruses.
Rhabdoviruses are enveloped and have helical capsid symmetry. These viruses
have a bullet-shaped morphology that is unique for this family. The genome is a
single-stranded negatine sense RNA.
The rabiesvirus is the only medically important species of rhabdoviruses. This is
the cause of rabies: an acute lethal infection of the central nervous system.
An epidemiologically important source of rabiesvirus is the sliva of the infected
animals. Rabiesvirus has a wide animal reservoir (e.g., foxes, skunks, racoons,
vampire bats). But, the principal reservoir for rabies in most of the world is the dog.
1. The first protective measure of major importance is local treatment of the wound.
Washing the wound with soap and water, detergent, or another substance that
inactivates the virus should be done immediately.
2. Postexposure immunoprophylaxis consists of the local infiltration and parenteral
administration of human rabies immunoglobulins and a coursr of rabies vaccin.
Rabies vaccin for human use contains inactivated rabies virus.
Picornaviridae is one of the largest families of viruses and includes some of the
most important human and animal viruses. They are smal (28-30 nm in diameter),
icosahedral, non-enveloped and have non-segmented single-stranded positive sense
RNA.
The family has more than 230 members that are divided into five genera:
Enterovirus, Rhinovirus, Heparnavirus, Cardiovirus and Aphthovirus.
Enteroviruses
Enterovirus Infections
Rhinoviruses
Rhinoviruses are the most important cause of the common cold and upper
respiratory tract infections. More than 100 serotypes of rhinovirus have been
identified.
Epidemiology
Rhinoviruses cause at least half of all upper respiratory tract infections. Other
agents that cause the symptoms of the common cold are enteroviruses, adenoviruses,
and parainfluenza viruses. Rhinoviruses can be transmitted by two mechanisms, as
aerosoles and on fomites (e.g., by hands or on contaminated inanimate objects).
Hands appear to be the major vector, and direct person-to-person contact is the
predominant mode of spread. These nonenveloped viruses are extremely stable and
can survive on such objects for many hours.
Clinical Syndromes
Common cold symptoms caused by rhinoviruses cannot readily be distinguished
from those caused by other viral respiratory pathogens. An upper respiratory tract
infection usually begins with sneezing, which is soon followed by rhinorrhea (runny
nose), nasal obstruction, mild sore throat, headache, and malaise. The illness peaks in
3 to 4 days, but the cough and nasal symptoms may persist for 7 to 10 days or longer.
Fever and rigors sometimes accompany rhinovirus infections. Otitis media in children
and sinusitis in adults are common complication.
Laboratory Diagnosis
The clinical syndrome of the common cold is usually so characteristic that
laboratory diagnosis is unnecessary.
HEPATITIS VIRUSES
Acute hepatitis is a common disease; most are due to virus infections. Viruses that
are primarily hepatotropic are frequently referred to as hepatitis viruses. There at least
six hepatitis viruses pathogenic for humans; they are hepatitis A, B, C, D, E and G
viruses. Although the target organ for each of these viruses is the liver, they differ
greatly in their structure, mode of replication, and the mode of tranmission and in the
course of the disease they cause.
Each of the hepatitis viruses infects and damages the liver, causing the classic
icteric symptoms of jaundice and the release of liver enzymes. These viruses are
readily spread because infected people are contagious before, or even without,
showing symptoms.
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Hepatitis A virus causes infectious hepatitis and is spread by the fecal-oral rout and
occurs mainly in children and young adults. This virus is the member of picornavirus
family and renamed enterovirus 72. It has the structural characteristic of a
picornavirus. There is only one serotype of HAV.
Diagnosis. This is made by serology (ELISA) testing for specific HAV IgM (acute
infection) or IgG (immune status).
Treatment. Symptomatic
Structure of The Vırus. HBV is the member of the hepadnavirus, a small (42 nm
in diameter) family of DNA viruses. Hepatitis B is an enveloped virus with
icosahedral capsid symmetry. The genome is a circular, double-stranded DNA,
encodes a reverse transcriptase, and replicates through an RNA intermediate. The
nucleocapsid of HBV also includes a DNA polymerase and a protein kinase
surrounded by the core antigen (HbcAg) and the hepatitis HBe antigen.
HBcAg is the major component of the nucleocapsid. HbeAg is a cleavage product
of the core antigen found on infected cells or free in serum.
The surface antigen (HbsAg) originally termed the Australia antigen, is found in
the envelope. The HbsAg elicits protective immunity
Replication of The Virus. On penetration into the cell, the nucleocapsid and
genome are delivered to the nucleus. The partial strand of the genome is completed to
form a complete double-strand DNA circule and then transcribed into individual
mRNAs by the host DNA-dependent RNA polymerase. A full-sized mRNA transcript
is also synthesized as a template for replication of the genome.
As with the retroviruses, HBV uses a reverse transcriptase to synthesize a
complementary DNA (cDNA) from the full-seized positive strand RNA. Unlike the
retrovirus, the cDNA is the negative strand of the virion rather than an intermediate
from the genome to be integrated. The RNA template and polymerase are
encapsulated within the nucleocapsid, and negative –starnd DNA synthesis occurs.
The RNA is degraded as the DNA is synthesized. The positive-strand DNA is then
synthesized from the negative DNA template as long as substrates are available.
Laboratory Diagnosis. The initial diagnosis of hepatitis can be made from the
clinical symptoms and the presence of liver enzymes in the blood. However,
identification of the specific viral agent requires quantitation of the HBsAg and
evaluation of the antibody response to HBsAg and HBcAg.
HBsAg, HBcAg, and HbeAg are secreted into the blood during virus replication.
The HbsAg and HbcAg are particulate, whereas the HbeAg is hidden, within the
virion. No test is readily available for HbcAg, but HbsAg and HbeAg can be detected
in clinical laboratory assays. Detection of HbeAg is the best correlate to the presence
of active infection.
Antibodies are produced against HBcAg and HbsAg, but so much HbsAg is
produced during the symptomatic phase of infection that the antibody is complexed
and not detectable. Indication of recent acute infection, especially during the period
when neither HbsAg nor anti-HBs can be detected, is best established by measurment
of IgM anti-HBc.
Other measures.
1. Universal testing of donated blood products.
2. Use of condoms.
3. Use of clean needles and other skin-piercing equipment.
4. Carriers of HbsAg may be restricted from certain occupations. The disease is
notifiable.
Chronic infection with delta agent can occur in those individuals with chronic
HBV.
Prevention and Control. Control measures are the same as for hepatitis B,
although no specific immunoglobulin or vaccine exists. Controling the spraed of
hepatitis B prevents the occurrence of hepatitis D.
There is still another type of non-A non-B hepatitis that occurs primarily in less
developed countries and in epidemics. This type of infections is enterically
transmitted and is caused by a nonenveloped RNA virus about 30 nm in diameter. It
does not cross react serologically with hepatitis A, B, C, or D viruses. It has
tentatively named hepatitis E virus and related to caliciviruses.
Pathogenesis may be similar to hepatitis A. Diagnosis is based on epidemiology
and exclusion of other hepatitis. In research laboratories, immunoelectron
microscopic examination of feces is useful.
The mortality rate associated with HEV disease is 1% to 2%, approximately 10
times that associated with HAV disease. HEV infection is especially serious in
pregnant women (mortality rate of approximately 20%).
Specific antiviral treatment is not available. Prevention depends on strategy for
minimizing fecal-oral transmission of infectious agents.
has risen exponentially, and it is now estimated that over 40 million cases have
occurred worldwide. Two-thirds of AIDS cases have occurred in Africa.
The HIV mutates at very high freguency during its replication. Presumably, the
mutations occur during the reveres transcription of the viral genome to proviral DNA.
Therefore, HIV isolates are very heterogenous genomically, serologically, and
biologically. The coexistence of genomically distinct HIV in the same patient has
been documented. Mutation may effect the env gene (coding for gp 120) and
produces mutants that are poorly neutralized by existing neutralizing antibody.
Virus isolates may differ in cell tropism. For examples, some isolates grow chiefly
in CD4 T cells, others grow chiefly in macrophages, and still others grow in both
CD4 T cells and macrophages. Virus isolates may also vary in their ability to
productively infect cells.
Replication
1. Adsorption to virus-specific receptor on CD4 cells; and receptor-mediated
endocytosis.
2. Synthesis of double-stranded complementary DNA (cDNA) by viral reveres
transcriptase.
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The principal cell receptor for HIV attachment is the CD4 molecul. As expected,
cells rich in CD4 molecules, such as CD4 T-lymphocytes and subpopulation of
macrophages, are the principal target cells of HIV. HIV-induced immunosuppression
(AIDS) results from a reduction in the number of CD4 T cells, which decimates the
helper and delayed-type hypersensitivity functions of the immune response.
During vaginal or anal sexual intercourse, HIV infects Langerhans dendritic cells
in the epithelium, and these can then travel to lymph nodes. Anal sex may be of
greater risk than other routes of infection. On injection of virus into blood, the virus is
likely to infect dendritic and other monocyte-macrophage lineage cells. The virus
reaches the lymph node within 2 days of infection, and there the CD4 T cells are
infected. Macrophages express lesser amounts of CD4 than T cells but can still be
infected with HIV. The virus does not kill the cells but may alter their function.
Monocytes and macrophages are probably the major reservoirs and means of
distribution for HIV.
After infection of these cells, the virus replicates but rapidly established latency.
The virus may remain latent for long periods, but when activated in CD4 T cells, the
virus kills the cell. Reductions in the numbers of CD4 T cells may result from HIV-
induced cytolysis and cytotoxic T-cell immune cytolysis.
HIV inducess several cytopathic effects that may kill the cell. These include
accumulation of nonintegrated circular DNA copies of the genome, increased
permeability of the plasma membrane, and syncytia formation (formation of
multinucleated cells).
Changes in cell function also occur in CD4 T cells latently infected with HIV. HIV
infection can reduce the expression of CD4 antigens and production of interleukin
(IL-2), reducing the ability of the helper cell to respond to antigen and activate other
T cells.
The immune response to HIV restricts viral infection but contributes to
pathogenesis. Neutralizing antibodies are generated against gp120 and participate in
antibody-coated virus is infectious, however, and is taken up by macrophages. CD8 T
cells can kill infected cells by direct cytotoxic action and by producing suppressive
factors that restrict viral replication. However, CD8 T cells require activation by CD4
T cells, CD8 T-cell number decreases with CD4 T-cell number, and their reduction
correlates with disease progression to AIDS.
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HIV has several ways of escaping immune control. Most significant are the virus’s
ability to undergo mutation, alter its antigenicity and escape antibody clearance, and
the targeted killing of the CD4 T cell.
Clinical Manifestations
Latent phase. The latent phase of the HIV infection may last from 18 months to 15
years or more, with an average of about 8 years. For much of this time the individual
is well, not unduly susceptible to infections and recovers apparently normally from
common and seasonal infections. The total CD4 T helper cell population slowly
declines, CD4 helper function is increasingly impaired.
Symptomatic HIV infection. A sudden increase in the loss of CD4 cells heralds the
end of the latent phase. Clinical evidence of immunodeficiency includes generalized
infections with:
. Troublesome bacterial infections, most importantly pneumococcal
(Mycobacteria) and Salmonella infections.
. Viral infections, herpes zoster is common and cytomegalovirus disease.
. Fungal infections. Oral and genital candidiasis.
. Protozoal infections. Pneumocystis and toxoplasma
And tumours. Kaposi’s sarcoma, lymphomas.
Epidemiology
The principal sources of HIV are the blood and genital secretions of HIV infected
persons. Other body secretions or fluids may contain HIV in low concentrations and
are probably unimportant in transmission. Three main modes of transmission have
been recognized:
1. Sexual contact with an infected person.
2. Inoculation of blood or blood products contaminated by HIV (mainly through
needle sharing among IV drug users.
3. Perinatal transmission from an infected women to her fetus or infant. No other
mode of transmission has been firmly established.
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Laboratory Diagnosis
2. Serology
ELISA or agglutination procedures are used for routine screening. The ELISA test,
however, can yield false-positive results and will not detect a recent infection. More
specific procedures, such as the Western blot analysis, are subsequently used to
confirm seropositive results. The Western blot assay, on the other hand, determines
the presence of antibody to the viral antigens (p24 or p31) and glycoproteins (gp41
and gp120). HIV antibody may develope slowly, taking 4 to 8 weeks in most patients;
however it may take 6 months or more in as much as 5% of those infected.
3. Immunology
The status of an HIV infection can be implied from an analysis of the T-cell
subsets. The absolute number of CD4 lymphocytes and the ratio of CD4 to CD8 are
abnormally low im HIV-infected people. The particular concentration of CD4
lymphocytes identifies the stage of AIDS.
Treatment
The most effective control measure is the use of condoms. Other preventive
measures include screening of blood products, needle exchange schemes, and
voluntary testing of those in high-risk categories.
Viroids are small molecules of single-stranded RNA less than 0.5 kb in size and
are capable of replicating in sesceptible cells. The RNA have regions of internal
complementarity allows for self-anneal by internal base pairing, thereby yielding
stable rolike. It is not clear if viroids code for viroid specific proteins. Since the host
does not have RNA replicase and the viroid genome is too small to code for such an
enzyme, how viroids multiply remains unresolved. Viroids are conceptually but not
medically important since all viroids identified thus far are plant pathogens.
Virusoids are similar to viroids except that the virusoids is encapsulated in an RNA
virus and that the virusoid needs helper function from the RNA virus for replication.
Various areas of the body normally support a microbiota even in the state
ordinarily called health. The oral cavity supports one of the most concentrated and
varied of such microbial populations, with main foci on the dorsum of the tongue,
about gingival sulcus, and in coronal dental plaque. It has been estimated that about
400 different microbial species are capable colonizing the dentate oral cavity and that
any individual may harbor over 150 different species.
In utero, however, the fetus is normally germ-free. During birth, a child is
presumably inoculated with the normal flora of the mother’s genital tract, that is,
some or all of the following: lactobacilli, corynebacterium, micrococci, coliforms,
alfa-, beta-, and anaerobic streptococci, yeasts, protozoa, and possibly viruses. With
the exception of the Streptococcus salivarius, which can be cultured with some
degree of regularity, most of these organisms are found sporadically, and not in high
numbers.
The oral flora is exclusively of aerobic type during the period preceding dentition,
and that, thereafter, anaerobic forms definitely characterizes the microbial phases of
the oral cavity. The early period is dominated by facultative species, to which are
added gradually the various obligate anaerobes, but numerically the facultative types
generally dominated at all ages. The presence of teeth would provide regions for the
growth of organisms adapted to the environmental conditions these structures
provide; for example, Streptococcus mutans seems to colonize preferentially in
plaque.
The oral cavity may be broadly subdivided into three major components: the teeth,
the supporting structures of the teeth (periodontium), and other intraoral structures,
including the lips, tongue, floor of the mouth, buccal mucosa, palate,
temporomandibular joint, fauces, and the tonsils. This lecture focuses on the teeth and
periodontium.
Teeth
teeth usually begins between 6 and 71/2 months of age with the lower central incisors.
The second molars are the last deciduous teeth to erupt, occurring between 20 and 24
months of age. Along with eruption of the deciduous teeth is an ever-increasing
complex of bacterial microflora.
The permanent teeth begin erupting between 6 and 7 years of age; the final teeth,
third molars “wisdom teeth,” erupt between 17 and 21 years of age.
Teeth can be subdivided into four basic substructures; enamel, dentin, cementum,
and pulp (Fig. 37).
Dental enamel is the hardest material in the body and consists nearly entirely of
inorganic salts (97%). This material covers the crown of the tooth and is usually
resistant to abrasive wear. Dental caries begins when the demineralization of enamel
occurs as result of acid production through the plaque constituents’ metabolism of
sugars.
Dentin, which is approximately 65% to 70% inorganic salts, is the calcified tissue
that forms the main structure of the tooth. Dentin is the second structure subjected to
dental caries. Pain is generally not perceived by patients unless dentin has been
destroyed by the caries process.
The cementum is thin and covers the roots of the teeth. One function of cementum
is to incorporate collagenous periodontal ligament fibers and thus mediate the
connection between teeth and alveolar bone. The cementum may also be colonized by
bacteria, and dental caries may result (root caries).
Dental pulp occupies the central area of the crown of the tooth and the roots. It is
soft tissue, primarily a collagen mass with a network of blood vascular and nervous
systems, that remains from the formative organ dentin. If dental caries continues
through the enamel, cementum, and dentin, the pulp may be invaded.
Periodontium
The periodontium consists of the investing and supporting tissues of the tooth
(gingiva, periodontal ligament, cementum, and alveolar bone). It has been divided
into two parts:
The gingiva, whose main function is protection of the underlying tissues, and the
attachment apparatus, composed of the periodontal ligament, cementum, and alveolar
bone. It is divided anatomically into marginal, attached, and interdental areas.
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. The marginal, or unattached, gingiva is the terminal edge or border of the gingiva
surrounding the teeth collarlike fashion.
. The attached gingiva is continuous with the marginal gingiva. It is firm and
tightly bound to the alveolar bone.
. The interdental gingiva occupies the gingival embrasure, which is the
interproximal space beneath the area of tooth contact.
The cementum is considered a part of the periodontium because, with the bone, it
serves as the support for the fibers of the periodontal ligament.
Tooth-Associated Materials
Food Debris
Food debris is retained in the mouth after mastication of food. This debris, unless
wedged between the teeth or inside the periodontal-tooth interface, is usually
removed by saliva or oral musculature action.
Acquired Pellicle
Acquired pellicle is a very thin (0.1 to 0.8 µm), primarily protein film that forms on
surfaces of the teeth. This pellicle is derived from components of saliva and
crevicular fluid as well as bacterial and host tissue cell products and debris. It is
acquired very soon (within minutes) after the teeth are cleaned. Acquired pellicle is
considered the first stage in the formation of dental plaque, since bacterial adherence
followed by colonization quickly ensues.
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Materia Alba
Materia alba refers to soft accumulations of bacteria and tissue cells that lack the
organized structure of dental plaque and are easily displaced with a water spray.
Bacterial Plaque
The normal flora of the oral cavity is responsible for a large proportion of oral
infections (e.g. caries, abscesses, candidiasis). Lesions in the mouth can also be
associated with generalized infections (e.g. Koplik spots in measles).
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DENTAL CARIES
. The Chemicoparasitic or Acidogenic Theory. This theory has been the most
popular over the years and is probably the one most widely accepted today. It is
generally agreed that dental caries is caused by acid resulting from the action of
microorganisms on carbonhydrates. It is characterized by a decalcification of the
inorganic portion and is accompanied or followed by a disintegration of the organic
substance of the tooth.
A number of microorganisms can produce enough acid to decalcify tooth structure,
particularly aciduric streptococci, lactobacilli, diphtheroids, yeasts, staphylococci,
and certain strains of sarcinae. The most frequently isolated bacteria associated
with dental caries is Streptococcus mutans. Their numbers increase with initiation
of dental caries and decrease when caries are treated, exhibiting a strongn correlation
with this disease process. Streptococcus mutans produces an enzyme dextran-sucrase,
which converts the sucrose of food to dextrin, and dextrin combines with salivary
proteins to create a sticky, colorless film (plaque) on tooth surfaces. Plaque provides
the haven for the activities of Lactobacilli and these produce of lactic acid, which
attacks the enamel by decalcifying it.
Interestingly, S.mutans is not present in the oral cavity of infants at birth and can be
detected only after the primary teeth begin to erupt. The most common source of
transmission of the bacteria is from the mother to the child.
Dental caries extends through the enamel and into the dentin where the carious
lesion must be restored. Without appropriate treatment at this time, the carious lesion
may furthere extend through the dentin and into the dental pulp region. Sensitivity to
heat and cold or sensitivity to percussion are possible symptoms related to extension
of the caries to the pulp.
After bacterial involvment of the pulpal regions during caries pathogenesis, a
periapical lesion (granuloma or abscess) may develop at the tip of the root. Root
canal therapy (endodontic therapy) is even more critical at this time and includes
mechanical removal and debridement of the pulpal contents of the tooth. When this is
performed, the periapical lesion frequently resolves without the use of antibiotic
therapy.
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PERIODONTAL DISEASES
Periodontal infections cause the most common diseases of the periodontium and
are among the most common of all diseases in humans. The periodontal diseases can
be broadly subdivided into two main categories: gingivitis and periodontitis.
GINGIVITIS
Gingivitis is an inflamation involving only the gingival tissues next to the tooth.
The primary etiologic agent of gingivitis is bacterial plaque. This forms the basis for
a classification of gingivitis, with “plaque-associated” gingivitis being the most
common form. The primary bacteria involved in gingivitis is hemolytic streptococci.
Other modifying secondary etiologic factors permit classification of other forms of
gingivitis. Acute necrotizing ulcerative gingivitis (ANUG), commonly known as
“trench-mouth,” “Vincent’s infection,” has several secondary etiologic factors and
constitutes another form of gingivitis. Two microorganisms, Borrelia vincentii and
Fusobacterium nucleatum, are generaly believed to be responsible for the disease.
Because many of these organisms are normal inhabitants of the oral cavity, smears
are of little value in establishing a diagnosis.
Unlike conventional periodontal disease, ANUG is most often observed in young
adults in their early twenties. It is the only periodontal infection in which bacterial
invasion of the gingival tissue has been observed.
The clinical features of ANUG are frequently seen by the practicing physician as
periodontal tissues with gingival craters covered by a grayish-white
pseudomembrane. Other clinical manifestations of the disease include inflamed,
painful, bleeding gingival tissue, poor appetite, fever as high as 40ºC, general
malaise, and a fetid odor.
Although once thought to be communicable, the disease connot be transmitted
solely by the transfer of organisms from one individual to another. Rather, it is
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PERIODONTITIS
In the oral cavity, bacteria colonize and accumulate principally in three regions: on
the dorsum of the tongue, on areas of the teeth not cleaned by mastications, and on
the teeth and gingivae in the vicinity of the gingival crevice or sulcus area.
Samples from the healthy gingival sulcus contain relatively few (10 3 to 106)
cultivable organisms, predominantly consisting of gram-positive facultative species
and members of the genera Streptococcus and Actinomyces (e.g., S.sanguis, S.mitis,
A.naeslundii, and A.viscosus). Small proportions of gram-negative species are also
found, most frequently Prevotella intermedia, Fusobacterium nucleatum, and
Capnocytophaga, Neisseria, and Veillonella spp. Microscopic studies indicate that a
few spirochetes and motile rods also may be found.
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On the basis of these criteria, several species have been related to the etiology
of destructive periodontal diseases, of which A. actinomycetemcomitans (A.a) and
P. gingivalis have the strongest association. A. actinomycetemcomitans is the most
important microorganism in juvenile periodontitis, while P. gingivalis is considered to
be associated with chronic (adult) periodontitis and refractory periodontitis. In low
numbers, Prevotella intermedia, and Prevotella nigrescence have been found in
periodontally healthy subjects, but they may also be associated with the development
of periodontitis. Furthermore, B. forsythus is found more frequently in periodontal
patients, and it is levels are related to probing depth and periodontal breakdown.
Other bacteria species with periodontal destruction include Fusobacterium
nucleatum, Campylobacter rectus, P. micros, Treponema denticola, and Treponema
vincentii. Like P. intermedia, these species are probably opportunistic pathogens with
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relatively low pathogenic potential and have to colonize the subgingival area for
longer periods of time at elevated levels to be able affect the periodontium.
Types of Periodontitis
The disease periodontitis can be subclassified into the following three major types
based on clinical, radiographic, historical, and laboratory characteristics.
Chronic Periodontitis
Aggressive Periodontitis
Aggressive periodontitis differs from the chronic form primarily by the rapid rate
of disease progression seen in an otherwise healthy individual, an absence of large
accumulations of plaque and calculus, and a family history of aggressive disease
suggestive of a genetic trait. This form of periodontitis was previously classified as
early onset periodontitis. The disease may be localized or generalized and rapidly
progressive periodontitis. Aggressive forms of periodontitis usually affect young
individuals at or after puberty and may be observed during the second and third
dicated of life (i.e., 10 to 30 years of age).
The microbiota associated with localized aggressive periodontitis is predominantly
composed of gram-negative, capnophilic, and anaerobic rods. A.
actinomycetemcomitans is generally accepted as the primary etiologic agent in most,
but not all, cases of localized aggressive periodontitis. Other organisms found in
significant levels include P. gingivalis, E. corrodense, C. rectus, F. nucleatum, B.
capillus, Eubacterium brachy and Capnocytophaga spp. and spirochetes.
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Herpesviruses, includind EBV-1 and HCMV, also have been associated with localized
aggressive periodontitis.
Oral Syphilis
The primary lesion of acquired syphilis is the chancre, which is most common
periorally on the lip or corners of the mouth and occurs approximately 3 weeks after
contact with an infected lesion. Clinically, chancres usually present as painless
ulcerations. Lymphadenopathy with tenderness is also noted. The chancre heals
spontaneously in about a month. Swabs of the ulcer cannot be relied upon for
diagnosis as non-pathogenic spiral treponemas may be present in the normal flora.
Diagnosis is confirmed serologically. In secondary syphilis ulcers may develop on
skin and mucous membranes, including the lips, tongue and palate.
The tertiary lesion of acquired syphilis is the gumma. Unlike the other lesions of
syphilis, the gumma is not seen in proximity to the initial infection and, in fact, is
noted 2 to 10 years later. Clinically, the gumma is rare in the mouth but, when
present, is seen as a deep penetrating ulcer, often involving the palate or tongue.
Unlike the primary and secondary forms of syphilis, the gumma is not infectious.
Candidiasis
Of the fungal infections that affect the mouth, candidiasis is the most common.
Approximately 50% of the population has Candida albicans in the normal oral flora.
Usually this organism is of no clinical significance. If changes ocuur in the oral
enviroment, however, candidal organisms can proliferate and cause infection.
Candidiasis is most frequent in patients at either age extreme; in newborns the
infection is called thrush. It has been reported that fungal proliferation is seen
frequently under the palate of maxillary prostheses. Any patient whose marrow status
changes because of diseases such as aplastic anemia or drugs is at high risk to
develop candidiasis. Similarly, patients reciving immunosuppressive therapy such as
asteroids or debilitated individuals such as patients with diabetes may develop fungal
infections of this type. Mucocutaneous candidiasis is one of the most frequent signs
of symptomatic HIV infection. Prolonged use of antibiotics, particularly broad-
spectrum agents, may produce candidiasis.
Clinically, candidiasis has a wide range of manifestations. Patients may be totally
asymptomatic or complain of pain or burning or of having a coated feeling in the
mouth. On examination, the clinician may note raised, curdy white areas that often
appear to lump in heaps. The tongue may take on a coated white appearance. The
borders of the white areas may be erythromatous. The white areas can usually be
scraped off with firm pressure using a wooden tongue blade, leaving a raw bleeding
surface. A smear of the material reveals the presence of hyphae when observed
microscopically.
Treatment is made by nistatin oral suspension. Candidiasis beneath dentures can be
traeted with nistatin ointment applied to the prosthesis. Systemic medications for
mucosal candidiasis include ketoconazole tablets.
Dental Abscesses
Actinomycosis
Actinomyces israelii may cause infection of the mandible and associated tissue.
Sinus formation may occur, with “sulphur granule” discharge which contains clumps
of the organism. Diagnosis is based on history, clinical presentation, and culture of
drainage. The infection is usually responsive to a prolonged antibiotic course;
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penicillin is the drug of choice. Surgical debridement of the infected region is often
helpful.
Herpetic Stomatitis
Herpangina
Characteristics
Fungi are eukaryotic organisms; they are distinct from plants in not containing
chlorophyll. Fungi are macroscopic (mushrooms) or microscopic (moulds and
yeasts). Only a few species cause human disease. Microscopic fungi are non-motile;
they may grow as single cells (yeasts) or filamentous structures (mycelia), some of
which may be branched.
Classification
Fungi are classified according to their method of sexual reproduction. Those that
do not reproduce sexually are called ‘fungi imperfecti’; those that reproduce sexually
may be self-fertile or require strains of an opposite type to allow sexual fusion to
occur.
Four groups of fungi cause human disease.
• Yeasts: have round or oval cells that multiply by budding, e.g. Cryptococcus
neoformans.
• Yeast-like fungi: grow predominantly as yeasts that can bud. They may also form
chains of elongated filamentous cells called pseudohyphae, e.g. Candida albicans.
• Filamentous fungi: grow as filaments (hyphae) and produce an intertwined
network called a mycelium. They produce asexual spores (conidia), which may be
single or multi-celled. Conidia are produced in long chains on an aerial hyphae
(condiophore): e.g. Aspergillus, Trichophyton and Zygomycetes (including
Mucor).
• Dimorphic fungi: have two forms of growth: filamentous at 22 C (saprophytic
phase) and yeast-like at 37 C (parasitic phase); examples include Blastomyces,
Coccidioides, Histoplasma capsulatum.
Fungal Infections
Fungal infections can be divided into two groups, superficial and deep mycoses.
Most deep mycoses are opportunist infections occurring in immunocompromised
patients.
Superficial Mycoses
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Laboratory diagnosis
• Direct microscopy can be made of clinical material, including skin scrapings and
sputum; the characteristic morphology facilitates identification.
• Culture: fungi can be isolated on most routine media, but may require a prolonged
incubation. Antibiotic-containing selective media (e.g. Sabouraud’s glucose agar),
which inhibit bacterial growth, are often used. Incubation at both 37 C and 28 C
facilitates the isolation of common filamentous fungi and yeasts.
• Serology: serological tests are available for the diagnosis of some fungal
infections (e.g. candida and aspergillus), but these lack spesificity and sensitivity.
Cryptococcus neoformans
Deep Mycoses
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Candida
• The genus Candida contains a number of species, including C. albicans (the most
frequently isolated pathogen), C. parapsilosis and C. tropicalis. C.
albicans is a commensal of the mouth and gastrointestinal tract.
• Superficial candida infections are common and include vaginal and oral
candidiasis (thrush), skin and nail infections. These infections may arise as a
complication of antibiotic therapy which reduces temporarily the bacterial flora.
• Invasive candida infections may involve the gastrointestinal tract (e.g.
oesophagus), lungs and urinary tract. Candidaemia may result in abscesses in
various organs (e.g. brain, liver). These infections occur primarily in
immunocompromised patients.
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• Candida can also colonize prosthetic materials, e.g. intravascular catheters and
peritoneal dialysis cannulae, resulting in septicaemia and peritonitis, respectively.
Candida is a rare cause of endocarditis.
Malassezia furfur
Aspergillus
Dermatophytes
The dermatophytes are a group of related filamentous fungi, also referred to as the
ringworm fungi which infect skin and related structures. Three clinically important
genera have been described.
Epidemiology. The natural habitat is man, animals or soil; human infection results
from spread from any of these reservoirs. Dermatophyte infections are found
worldwide with different species predominating in various climates.
Laboratory diagnosis
• Skin scrapings, hair or nail clippings from active lesions are examined
microscopically in 30% potassium hydroxide on a glass slide; the presence of
hyphae confirms the diagnosis. Occasionally, the dermatophyte species can be
identified by typical morphology.
• Samples can be cultured on Sabouraud’s medium at room temperature.Subsequent
species identification is based on growth rate, colony appearance and microscopic
morphology.
• Infected hair may fluoresce under ultraviolet light (Wood’s light) and is
characteristic of certain infections, e.g. M. canis.
Treatment. Depends on the site and severity of infection. Options include topical
imidazoles (e.g. clotrimazole, miconazole); oral griseofulvin, itraconazole or
terbinafine.
Mucormycosis
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Epidemiology. The Mucorales are found worldwide, in soil and decaying organic
matter. Infections occur primarily in the immunocompromised. As with aspergillus,
hospital outbreaks of infection have occurred in association with building work.
Infection
• Pulmonary: an often fatal infection of immunocompromised patients may result in
disseminated infection (e.g. brain, liver, gastrointestinal tract).
• Rhinocerebral: an infection of the nasal sinuses may spread rapidly to involve the
face, orbit, and brain. It occurs particularly in uncontrolled diabetes mellitus and is
often fatal if treatment is delayed.
Coccidioides immitis
C. immitis is a dimorphic fungus found in soil in hot arid areas of south-west USA,
Central and South America.
Treatment. Amphotericin.
Blastomyces dermatitidis
Histoplasma capsulatum
Paracoccidioides brasiliensis
PARASITOLOGY
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Although the term “parasite” may be applied broadly to all infectious agents,
including bacteria, viruses, and fungi, it is traditionally reserved for parasitic
protozoa, helminths (worms), and arthropods. The classification of parasites used in
this chapter is simplified and uses common terminology.
CLASSIFICATION
Parasites are classified within the kingdom Animalia and are separated into two
subkingdoms, Protozoa and Metazoa. The subkingdom Protozoa comprises
unicellular animals in which all life functions occur in a single cell. The Metazoa are
multicellular animals in which life functions occur in cellular structures organized as
tissue and organ systems.
Protozoa
Metazoa
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The subkingdom Metazoa, which includes all animals that are not Protozoa, has
two groups of organisms of major importance in this text, the helminths (worms)
and the arthropods (crabs, insects, ticks, etc.).
DEFINITIONS
Below are definitions of terms that are used in describing parasite life cycles and
host-parasite interrelationships:
Definitive Host: A host that harbors the adult or sexual form of the parasite.
Intermediate Host: A host that harbors larval or asexual stages of the parasite.
Reservoir Host: A nonhuman host that can maintain the infection in nature in the
absence of human hosts.
Vector: An arthropod that is responsible for the transmission of parasite from host
to host. If the arthropod is not essential to the life cycle, it is a mechanical vector; if
it is essential, it is a biologic vector.
Symbiosis: The relationship between two dissimilar organisms that are adapted to
living together is called synbiosis and the associates are symbionts. The association
may be beneficial or harmful to either of the associates. Symbiosis may be divided
into categories:
• Mutualism: A symbiotic association that is beneficial to both parties.
• Commensalism: One of the associated organisms is benefits and the other is
neither benefits or harms.
• Parasitism: One organism (the parasite) benefits at the expense of the other (the
host). Organisms that live on or in the skin of their hosts are ectoparasites. This
relationship is an infestation. Most parasitic arthropods belong to this category.
Parasites of the digestive tract, extraintestinal organs and tissues, and those that are
intracellular within the host are referred to as endoparasites and produce infection,
irrespective of their size.
Fecal Examination
Fecal examination for ova and parasites is used for most intestinal parasites. In
clinical jargon, this is often referred to as “stool for O and P” when an order is written
to collect fecal specimens for parasitologic examination.
Preservation of fecal samples. For periods of several hours and sometimes longer
good preservation is obtained by refrigeration at 3 to 10˚C. For reliable and lasting
preservation, chemical fixation is required.
• Formalin. Eggs and larvae of helminths found in stool specimens are
satisfactorily fixed and preserved for later examination in 10% formalin heated to
65˚C (1 part of feces to 5-10 parts of 10% formalin).
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Permanent fecal film. To make a permanent, stained film of fresh feces for study of
intestinal protozoa, a representative fleck of the material is smeared as evenly as
possible in a thin film on a clean slide and immediately immersed in Schaudinn’s
solution.
Antigen detection systems are new and promising supplements to the microscopic
examination of stool. These systems rely on the use of antibodies to specific antigens
excreted in the stool of infected individuals. Antigen in the stool binds to antibody.
Enzyme technques that produce a visible colored product are used to detect the bound
antibody.
Blood Films
Next to feces, blood provides the most common medium for recovery of various
stages of animal parasites. From this source, diagnosis is routinely made of malaria,
African trypanosomiasis, and others. Giemsa stain is preferred for most parasitic
infections.
Thin films are made by drawing out a drop of blood on a microscope slide so that
the red cells from an even layer that is one cell thick. The film is then fixed and
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Thick films are made by stirring several drops of blood on a microscope slide into a
single circular area. The blood is allowed to dry. The slide is placed in water that
removes hemoglobin from the RBCs. The slide is then stained. This technique allows
the concentration of organisms in a small area for easier detection.
Serologic Tests
Serologic tests, which detect antibody to the parasite, are becoming increasingly
valuble as specific antigens are identified and used in sensitive tests, such as ELISA
and in immunoblotting methods.
PROTOZOA
AMOEBAE
The amebae are primitive, unicellular microorganisms, with a simple two stage life
cycle: the actively motile feeding stage (trophozoite) and the quiescent, resistant,
infective stage (cyst). Replication is accomplished by binary fission (splitting the
trophozoite) or by the development of numerous trophozoites within the mature
multinucleated cyst. Motility of amebae is accomplished by extension of a
pseudopod (“false foot”). The amebic trophozoites remain actively motile as long as
the enviroment is favorable. Cyst formation occurs under adverse conditions.
Most amebae found in man are commensal organisms (Entamoebae coli,
Entamoebae gingivalis, etc.). However, Entamoebae histolytica is an important
human pathogen. Some free-living amebae (Naegleria fowleri, Acanthamoebae
species) are present in worm, freshwater ponds or swimming pools and can be
opportunistic human pathogens, causing meningoencephalitis or keratitis.
E.histolytica has four distinct stages in its life cycle: trophozoite, precyst, cyst, and
metacyst. The stages commonly recognized in the feces are trophozoites and cysts;
only trophozoites are present in the tissues.
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The trophozoite in its natural habitat in the large intestine and in extraintestinal foci
generally varies from 12 to 30 μ in diameter. It is contain a single spherical nucleus
that has a small central karyosome and peripheral chromatin, which is evenly
distributed on the nuclear membrane in small granules. The cytoplasm is divided into
endoplasm, the granular portion that often contains vacules (with ingested RBCs),
and ectoplasm the portion that is clear.
The pseudopod is a protrusion of cytoplasm. Usally clear ectoplasm is extended in
a blunt or fingerlike projection followed by the flowing of endoplasm into the
pseudopod. This process is the method of locomotion in amebae.
The cyst is the resistant stage that is excreted in the stool. The mature cyst has a
refractile cyst wall and contains four nuclei that resemble the nuclei of the
trophozoites. Young cysts often contain deeply staining bundles of crystalline RNA
that are called chromatoidal bodies. Cysts range in size from 10 to 20 μ in diameter.
Pathogenesis
Following ingestion the cysts pass through the stomach, where exposure to gastric
acid stimulates release of the pathogenic trophozoite in the small intestine. The
trophozoites divide and produce necrosis and ulceration in the large intestine.
Invasion through the gut wall into the peritoneal cavity and blood stream spread to
other organs (primarily the liver) may occur. The cysts are excreted in the stool.
Trophozoites are found in the tissues in invasive disease. Cysts do not form in tissues.
Clinical Manifestations
Dysentery (acute amebic colitis) is associated with diarrhea containing blood and
mucus. Liver abscesses and, less commonly, lung and brain abscesses are found.
Laboratory Diagnosis
Microscopy of freshly passed stools for the presence of trophozoites and cysts, or
for trophozoites in abscesses. E.histolytica must be differentiated morphologically
from other intestinal protozoa. There are other species of Entamoeba that resemble
E.histolytica, such as the E.coli and E.gingivalis, which is found in human mouth.
Treatment
The treatment of amebiasis has two major components: the elimination of tissue
invading organisms and the elimination of organisms from the lumen of the intestine.
The drugs that are most commonly used for tissue invasion are metronidazole,
emetine and others. Drugs that are used to treat the luminal organisms are iodoquinol
and diloxanide furoate.
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FLAGELLATES
Giardia lamblia Stiles, 1915 has a cosmopolitan distribution and is common in both
warm and temparate climates.
Pathogenesis
Infection results from the ingestion of cysts from feces. Contaminated water is
frequent source and a few food borne outbreaks have been reported.
Cysts are ingested and gastric acid stimulates the release of trophozoites in the
small intestine, which then multiply by binary fission. Trophozoites attach to the
intestinal villi by a sucking disc. İnflammation of the epithelium may occur, but
systemic invasion is rare. Cyst formation occurs as the organisms move through
colon.
Clinical Manifestations
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Some infections may be asymptomatic. When symptoms occur, they may vary
from severe acute diarrhea to chronic mild diarrhea associated with flatulence,
abdominal discomfort. In some instances, significant intestinal malabsorption ocuurs.
Laboratory Diagnosis
Stool microscopy for cysts and trophozoites. If clinical suspicion is high and stool
examination is negative, duodenal aspiration or biopsy may be helpful.
Treatment. Metronidazole
This organism is found in the vagina and urethra in females and in the urethra,
seminal vesicles, and prostate in males.
Epidemiology
This parasite has worldwide distribution with sexual intercourse as the primary
mode of transmission. Occasionally, infections have been transmitted by fomites
(toilet articles, clothing), although this is limited by the ability of the trophozoite
form. Infants may be infected by passage through the mother’s infected birth canal.
Clinical Manifestations
Most infected women are asymptomatic or have a scant, watery vaginal discharge.
Vaginitis may occur with more extensive inflammation and erosion of the epithelial
lining, associated with itching, burning, and painful urination. Men are primarily
asymptomatic carriers who serve as a reservoir for infections in women.
Laboratory Diagnosis
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Treatment
Leishmania Species
When a sand fly bites an infected person or reservoir host, it sucks up parasitized
macrophages or temporarily free parasites (amastigotes) in the blood or tissue juices.
Soon after the amastigotes reach the midgut of the fly, they transform into the
flagellated promastigote forms, which after rapid multiplication transform into
infective promastigotes and migrate forward. From the foregut they are regurgiated or
otherwise introduced into the skin of the next individual when the sand fly takes
another blood meal.
Two stages in the life cycle are known: the amastigote in man and reservoir
mammals and the promastigote in the sand fly and in cultures. The amastigote is
spherical or subspherical and measures 2 to 5 μm in its greatest dimension. It lives
and reproduces by longitudinal binary fission in macrophages of skin, mucosa, lymph
nodes, and reticuloendothelial system. In preparations stained with Giemsa’s or
Wright’s stain, the cytoplasm is pale blue and the relatively large nucleus is red. In
the cytoplasm, usually lying in the medium line of the cell, is a deep red rodlike
structure called the kinetoplast; a delicate filament called the axoneme extends from
near the kinetoplast to the cell membrane.
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Visceral Leishmaniasis
Cutaneous Leishmaniasis
Mucocutaneous Leishmaniasis
Trypanosoma Species
Trypanosomes are haemoflagellates and live in the blood and tissue of human
hosts. The life cycle involves two hosts: blood-sucking insects and mammals. It
causes two diseases.
• African trypanosomiasis (sleeping sickness) caused by T.brucei gambiense and
T.brucei rhodesiense, and is transmitted by the tsetse fly.
• American trypanosomiasis (Chagas’ disease) caused by T.cruzi, and is transmitted
by the reduviid bug.
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The infective stage of the organism is the trypomastigote, present in the salivary
glands of transmitting tsetse flies. Trypomastigote varies greatly in length from 17 to
30 μm. It has an undulating membrane that runs the length of the organism. In the
long, slender forms, the undulating membrane terminates in a free flagellum at the
anterior end. There is a prominent nucleus in the midbody region. The kinetoplast is
found near the origin of the flagellum and undulating membrane at the posterior of
the organism.
The trypomastigotes enter the wound created by the fly bite and find their way into
blood and lymph node, eventually invading the central nerves system. Reproduction
of the trypomastigotes in blood, lymph, and spinal fluid is by binary fission.
These trypomastigotes in blood are then infective for biting tsetse flies, where
further reproduction occurs in the midgut. The organism then migrate to the salivary
glands where an epimastigote form (having a free flagellum but only a partial
undulating membrane) continues reproduction to the infective trypomastigote stage.
Tsetse become infective 4 to 6 weeks after feeding on blood from a diseased patient.
Clinical Manifestations
A nodule or chancre can form at the site of the bite, which usually resolves
spontaneously. The parasite enters the blood stream and results in lymphadenopathy,
irregular fever and myalgia. CNS involvment (sleeping sickness) may occur with
lethargy and encephalitis, leading to convulsions, coma and occasionally death.
Laboratory Diagnosis
Microscopy of thick and thin blood films, lymph node aspirates and cerebrospinal
fluid for trypomastigotes; serology (e.g., ELISA).
T.cruzi is found in North, central and South America. The life cycle is similar to
other trypanosomes, except when the infected bug bites humans, trypomastigotes are
released simultaneously in the faeces; these enter the wound following scratching.
Spread is via the lymphatic and blood systems and results in invasion of many
organs, including liver, heart, muscle and brain.
Within host cells, trypomastigotes transform into amastigote, which multiply by
binary fission and form either further amastigotes or trypomastigotes; the latter are
ingested by a feeding insect, multiply in the intestine and are then passed in the
faeces.
Chagas’ disease may be asymptomatic, acute or chronic. A painful nodule may
form at the bite. Acute infection results in high fever, erythematous rash, oedema and
myocarditis. Chronic disease may develop years after the initial infection. Organisms
proliferate in various organs, including the brain, spleen, liver, heart and lymph
nodes, resulting in lymphadenopathy, hepatosplenomegaly, myocarditis and
cardiomegaly. Granulomas and cysts may form in the brain. Cardiac disease is the
most common presentation, with congestive cardiac failure.
Laboratory diagnosis is made by microscopy of biopsies of affected tissues; thick
blood films for trypomastigotes in the acute phase; serological tests (e.g., ELISA).
Treatment is by nifurtimox.
COCCIDIA
Cryptosporidium parvum
These organisms are seen in the brush border of the intestinal epithelium of both
small and large bowel. Parasites have been seen in the gallbladder, tonsillar
epithelium, and bronchi in patients with AIDS.
The form of the organism excreted in the stool is called an oocyst. It contains four
sporozoites. Following ingestion, the sporozoites escape from the oocyst and
penetrate the intestinal epithelium, where they undergo asexual multiplication. The
organisms appear to be attached to the cell’s surface at the brush border, but are
actually under the cell membrane external to the cytoplasm. Sexual stage develop and
unite to form an oocyst that is shed in the feces. Some oocysts release sporozoites
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before they are excreted in the stool. These sporozoites enter intestinal epithelial cells
and continue the cycle (internal autoinfection).
Clinical Manifestations
Laboratory Diagnosis
Treatment
Isospora belli
I.belli has a life cycle similar to that of Cryptosporidium, but it is located deep
within the cytoplasm of the intestinal epithelial cell. The oocysts excreted in the stool.
Two spherical sporoblast are seen within the oocyst. The sporoblasts mature into
sporocysts, each containing four sporozoites.
Clinical Manifestations
Toxoplasma gondii
The definitive host for T.gondii is the domestic cat and other felids. The cat
acquires the infection by eating infected rodents or birds that contain tissue cysts or
by ingesting oocysts from the feces of other cats. In the cat, the parasite undergoes
reproduction in the intestinal epithelium. Sexual stages in the intestine result in the
production of oocysts that are excreted in the cat’s feces. The oocysts are infectious
for a wide variety of birds and mammals including humans and other cats. When
animals or humans ingest oocysts from cat feces, organisms escape from the oocyst
and develop into trophozoites called tachyzoites. Tachyzoites are elongated, crescent-
shaped organisms. The posterior end of the organism is more rounded than the
anterior end that contains the apical complex. There is a single nucleus. Tachyzoites
actively penetrate host cells, multiply intracellularly, and cause the rupture and death
of the host cell. While the tachyzoite is transiently free in the extracellular
enviroment, it is susceptible to lysis by antibody and complement. Once it has entered
a cell, it is protected from the lethal effects of humoral antibody. In macrophages, the
tachyzoites survive by inhibiting the fusion of lysosomes with the phagosome
containing the parasite. Eventually, some tachyzoites form cysts, more frequently in
brain, skeletal muscle, and heart. The organisms that develop within the cysts are
called bradyzoites.
Humans become infected from:
. ingestion of undercooked meat contaminated with trophozoites;
. ingestion of infected oocytes from the cat feces;
. transplacental transmission;
. cardiac transplantation; the recipient receives a heart containing toxoplasma cysts.
Clinical Manifestations
The two major categories of infection are acquired and conginital. Most acquired
infections are asymptomatic. When clinical evidence of infection is present, the most
common presentation of acquired infection is lymphodenopathy,usually cervical,
without associated symptoms. Some acquired infections may have systemic
manifestations, such as fever, headache, myalgia, lymphadenopathy, rash, and
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Laboratory Diagnosis
. Serology (e.g. ELISA): by rising IgG antibodies in paired sera, or IgM antibodies
to differentiate between active and previous infection.
. Histology: by examination of appropriate biopsies for cysts.
Treatment
Pneumocystis carinii
Historically, P.carinii has been placed with the sporozoites, but recent DNA studies
suggest that it may be a fungus. It is causes pneumonia in immunocompromised
individuals, such as AIDS patients, premature infants, malnourished children, patients
undergoing immunosuppressive therapy, and in children with congenital
immunodeficiencies.
P.carinii is seen in the pulmonary alveoli as thick-walled cysts with intracystic
bodies and as trophozoites.
The illness may be abrupt or insidious in onset. Fever and difficulty breathing are
common symptoms. Death from progressive pulmonary failure usually occurs in
untreated patients.
Diagnosis is made by direct examination of material, including bronchial biopsies,
washings or aspirates, and open lung biopsies. Parasites are identified by
histopathological stains or specific fluorescein-labelled antibodies. Serology may also
be used.
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Plasmodium spp.
All plasmodia share a common life cycle but with some important variations.
Anopheles mosquito introduces infective stages (sporozoites) into the human host
in its saliva during biting. The sporozoites liver parenchymal cells and multiply
asexually untile the parasites rupture from the cell. The released parasites infect
erythrocytes and begin a cycle of asexual reproduction (schizogony) within the
RBCs. This asexual cycle in the RBCs has the following stages:
1. The ring stage is the earliest form seen in the red cell. It consists of a ring of
cytoplasm containing a dot of nuclear material. The central area of the ring is a
vacule containing hemoglobin.
2. The trophozoite is the ameboid growing form of the organism before there is any
division of nuclear material. As the trophozoite develops, brownish or black
granular material appears within the cytoplasm. This malaria pigment is the iron-
containing, end product of hemoglobin metabolized by the parasite.
3. The early schizont stage begins with the first nuclear division. The cytoplasm of
the parasite has not yet undergo division.
4. The mature schizont (segmenter) has undergo complete division of the nuclear
material and the cytoplasm to form individual organisms called merozoites. When
the erythrocyte containing the mature schizont ruptures, the merozoites are
released and penetrate other RBCs to continue the cycle.
After several asexual cycles, some merozoites do not progress through asexual
development, but remain compact organisms that enlarge and differentiate into
either male (microgametocyte) or female (macrogametocyte) forms. These
gametocytes circulate in the blood and are unable to develop further unless taken
up by an anopheline mosquito during a blood meal. The sexual reproductive cycle
occurs in the mosquito’s digestive system. Sporozoites form, migrate to salivary
glands and are inoculated into a new host.
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P.vivax infects young RBCs. The parasite cause enlargement of the infected cell
and produce stainable alterations of the red cell membrane called Schüffner’s dots.
The trophozoites are very ameboid and the mature schizont contains 12-24
merozoites.
P.falciparum is able to infect RBCs of any age. The ring stage is small and delicate
and many have two chromatin (nuclear) dots. Gametocytes are cresent shaped. Only
rings and gametocytes are seen in the peripheral blood. The more advanced stages of
asexual development take place in venules where the infected red cells adhere to the
vascular endothelium.
P.malariae infects mature RBCs and these cells do not enlarge as the parasite
develop. The trophozoites tend to extend from one side of the red cell to the other in a
band. Mature schizonts contain 6-12 merozoites.
P.ovale infects young RBCs causes them to enlarge and produces Schüffner’s dots.
The infected cells are often ovoid and have ragged margins. The trophozoite remains
relatively compact, and the mature schizont usually has 4-12 merozoites.
Epidemiology
In tropical and subtropical areas plasmodia are dependent on correct conditions for
breeding of Anopheles mosquitos. P.falciparum is responsible for more than 80%
cases in tropical areas. In endemic areas, repeated infections/exposure result in
relative immunity and less severe disease. Victors to endemic areas are more severely
affected. Transmission via contaminated blood transfusions or needle-sharing can
ocuur rarely.
Laboratory Diagnosis
Thick and thin blood films are taken. The optimal stain for malaria parasites is
Giemsa; however, parasites should be easily recognized in films stained with
Wright’s stain. The typical morphology of the parasite within erythrocytes allows the
differentiation of Plasmodium species.
Treatment
Chloroquine is the treatment of choice for malaria due to P.vivax, P.ovale and
P.malariae. Supplementing treatment with primaquine is important to destroy the
liver hypnozoite stages of P.vivax and P.ovale.
Chloroquine is also the drug of choice for P.falciparum malaria, but chloroquine-
resistance is now common in some areas of the world. Alternative drugs include
quinine and the combination of pyrimethamine and sulphadoxine.
Prevention
CILIATES
HELMINTHS (WORMS)
NEMATODES
Ascaris lumbricoides
Clinical Manifestations/Complications.
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