BENUE STATE UNIVERSITY MAKURDI

DEPARTMENT OF BIOLOGICAL SCIENCES

LECTURE NOTE

MCB 405-MEDICAL MICROBIOLOGY

COURSE LECTURER:
Dr. Emmanuel Msugh MBAAWUAGA
B.Sc. Microbiology (Sokoto) M.Sc.Med. Microbiology (Jos)
Ph.D Med. Microbiology (Nigeria)

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 MECHANISM OF PATHOGENICITY

Pathogenicity is the ability of a disease causing organism to produce an infection in another

organism including human beings.

Disease causing organisms are known as pathogens. A pathogen can either be a primary or
an opportunistic pathogen;

Primary pathogen; produce disease in an otherwise healthy individual.

Opportunistic pathogen; is one which can produce in immune-compromised individual or the

organism is introduced into an unusual area or location, that is to say an organism can be a
normal flora on the skin but in the kidney or any other location, it can become pathogenic.

The methods that pathogens use to invade host defences and cause damage are called
Mechanisms of Pathogenicity. Normally, it is not in the interest of the pathogen to kill its host,
a pathogen that kill off its host will lose its habitation; hence, the pathogen-host relationship
evolved over time to give rise to a balanced pathogenicity.

This relationship can be illustrated in a case where Myxovirus was introduced in Australia
when the population of rabbits in the country became alarming, myxovirus which infects
rabbits was introduced to reduce the rabbit population but with time, rabbit population again
started increasing. The rabbits that survived the virus were found to be more resistant to the
original virus that was formally introduced; the viruses isolated from the surviving rabbits
were less virulent than the original virus.

The mechanism by which microorganisms cause disease generally follows one of several
patterns;

i. Pathogens can produce toxins that are then ingested: Here, there is no infection of the host,
i.e. the microorganism does not grow on/in the host, but produces toxins outside the host
that is then ingested. Typical example of such organism can be seen in food intoxication
caused by;Clostridium botulinum andStaphylococcus aureus .

ii. Colonization of the surface of the host followed by toxin production: Here, the organism
multiply to high number on a host surface and produce toxins that interferes with cell
functions e.g.Vibrio cholerae

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iii. Invasion of host tissue: Here, the microbe penetrates and multiplies within the host tissues
e.g. Mycobacterium tuberculosis, Streptococcus pyogens, Yersinia pestis and Salmonella
spp . To do this, the organism must penetrate the first line of defenses and breach the body’s
barrier.

iv. Invasion of host tissue followed by toxin production: In addition to penetrating and
multiplying within the host tissues, the organisms produce toxins e.g. Shigella dysentreae
and certain strains ofStreptococcus pyogens.

Specific Mechanisms of Pathogenicity

1. Establishment of Infection:

In order to cause disease, most pathogens must first adhere to the body surface then
multiply to high numbers to either produce enough toxins or invades. To establish infection,
pathogens follow this pattern;

a. Adherence;

The first lines of defenses are very effective in removing microorganism so a pathogen must
adhere to the host cell before establishing infection. However, not all attachment of
microorganism to cells results to disease. Bacteria use adhesins to bind to host cells.
Adhesins are often located at the tip of pili and pili that are used for attachment are referred
to as fimbrae. Adhesins can also be components of other surface structures such as
glycocalyx and some cell wall proteins. The surfaces receptor on some animal cells to which
the bacterial adhesins attach are usually glycoproteins or glycolipids and often the adhensin
will attach to the sugar component/molecule of the receptor e.g.Escherichia coli binds to the
mannose component of surface receptors in the intestine. The receptors and the animal
cells have not evolved to be used by the organism though; they serve specific purposes in the
host but are normally explored by microorganism to their own advantage.

The binding of adhesins to a surface receptor is highly specific however; some pathogenic
strains have additional adhesins that allow them to attach to cells of other tissues e. g E. coli
normally attach to intestine but E. coli that causes urinary tract infection generally produce a
type of pili called P-pili that allows them to attach to cells in the bladder.

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 b. Colonization;

To colonize the site populated by normal flora, pathogens must compete for space and
nutrients and overcome the toxic product of the normal flora such as fatty acids and
antimicrobial compounds called bacteriocins

They also must counter the body’s defenses aimed at protecting the surface such as
immunoglobulin-A antibodies. Mechanisms used by microorganisms to achieve these
include;

i. Rapid turnover of pili; it sheds antibodies that have bond by rapidly turning over of their pili.

ii. Antigenic variation which can alter the type of pili produced.

iii. Produced immunoglobulin-A proteases that cleaves the antibody molecules.

iv. Pathogens also produce iron binding molecules called Siderophore as well as used the iron
that has bond to host protein as nutrient. This is because iron is one of the very limiting
nutrients for bacteria.

c. Delivery of Infectors Molecules to Host Cells;

Once they have colonized, bacteria are able to deliver certain molecules directly to host cells
inducing changes in those cells. This is very common among the gastrointestinal pathogens.

Gram negative bacteria use type III secretion system to deliver protein to eukaryotic cells,
these structures resemble short flagella and functions as microscopic hypodermic needles
so both the secretion system and the transfer protein are often encoded by pathogenicity
islands ( a region in the chromosome where many virulence genes are located).

2. Invasion (Breaching Anatomical Barrier);

Pathogens that are able to pass the epithelia cell barrier will access the nutrient rich tissues
and multiply without competition. Barrier includes;

i. Penetration of skin: The most difficult barrier for a microbe to penetrate is the skin. Bacterial
pathogen invasion of the skin normally relys on trauma that destroys the integrity of the skin
e.g. Staphylococcus aureus access tissue via wound or they may rely on flea bites to inject

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 them;Yersinia pestis is injected through flea bite.

ii. Penetration of mucus membrane: the mucous membrane is the most common route of entry
for most pathogens. There are two general mechanism for invasion of the mucous
membrane; direct uptake by epithelia cell and exploitation of antigen-sampling process.

 Direct uptake by epithelia cell is when pathogens induce non-phagocytic epithelia cells to use
the process of endocytosis to take up bacteria cells. The pathogen attaches to a cell and
then triggers the cell to engulf it. Specialized proteins that cause changes in the host cell
cytoskeleton are delivered by the type III secretion system to induce uptake. The disruption
of the cytoskeleton causes morphological disturbances in the cell membrane called ruffling
and the bacteria sink into the ruffles, leading to their engulfment e.g. Salmonella spp and
Shigella spp normally penetrates the mucous membrane by this process.

 Exploitation of antigen sampling process: some bacteria exploit the sampling process used
by the immune system to gain access to deeper tissues. Several intestinal pathogens gain
access to tissues by way of M-cells of the Peyer’s patches in the intestine. This cell (Peyer’s
patches) samples intestinal contents and deliver them to macrophages in the lymphoid
tissues in a processes known as “Transcytosis”. Most microbes that are passed to the
macrophages in this way are destroyed but some have evolved mechanisms to avoid the
destruction e.g. Shigella spp passed by M-cells survive the phagocytic process of
macrophages by inducing apoptosis (programed cell death) in the cells, the bacteria then
adhere to specific receptors at the bed of epithelia cells and induce the epithelia cells to
engulf them, Mycobacterium tuberculosis also survive within macrophages that have not
been activated.

3. Avoiding Host Defenses:

Pathogens as a group have evolved a variety of mechanisms to avoid both innate and
adaptive defenses inside the host body, the mechanisms they used to achieve this includes;

i. Hiding within host cell;

Some bacteria invade some components of the host defenses by remaining inside host cells
out of the reach of phagocyte, complement and antibodies. Shigella and Listeria
monocytogenes are able to move from cell to cell using host cell actin and forming actin tail
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 that propelled the bacteria inside the cell.

ii. Avoiding killing by complement proteins;

There are three major outcome of activation used by complement system in getting rid of
bacteria. This could either be through Cytolyis, Opsonization or Chemotaxis.

The membrane activation complex (MAC) is most active against gram negative bacteria
since it is targeted towards lipoprotein and has little effect against Gram positive bacteria.
Because of the nature of their cell wall, some Gram negative bacteria can escape killing by
the complement protein and are said to be Serum resistant, they do this by capturing
complement regulatory proteins also known as complement control proteins (CCP) that
inactivate C3b (the major complement protein in the system) which inactivate the alternative
pathway of complement activation e.g.Neisseria gonorrheae andNeisseria menigititis .

iii. Avoiding Destruction by Phagocytes; Phagocytosis involves multiple steps

 Chemotaxis recognition and attachment

 Engulfment and fusion of the phagosome with the lysosome which leads to the destruction
and digestion of an invading microbe

Some pathogenic bacteria has evolved a variety of ways to avoid destructive effects of
phagocytes, they do this by;

a. Preventing Encounter with phagocytes: Some pathogens avoid the process of phagocytosis
by avoiding macrophages and neutrophils all together, one way they do this is by destroying
the complement component that attracts phagocytes, they attack C5a peptidase to degrade
C5a; a chemo-attractant which attracts macrophages and neutrophil to destroy bacteria
while C5b initiates membrane activation complex. Example of an organism that does this is
Streptococcus pyogenes . Another way is to kill the phagocytic cells as they arrive, this is
done using membrane damaging toxins which forms pores in their membrane. S. Pyogenes
produces membrane damaging toxins known as Streptolysin O.

b. Avoiding Recognition and Attachment: mechanism used to do these includes

 The use of capsules which can inactivate C3b preventing it from been an effective Opsonin

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 (substance that coats cells making it attractive to chemotaxis).

Opsonization is the coating of cells either by antibodies of C3b

 Binding complement control protein and preventing activation of the alternative complement
pathway as in serum resistant organisms e.g. Streptococcus pneumonieae. M-protein, a
component of the cell wall of S. Pyogenes acts in the same way as the capsule of S.
Pneumonieae above; inactivating C3b and binding complement regulatory proteins.

 They can also do this through the use of Fc receptor; Fc receptors are found on the surface
of Staphlococcus aureus referred to as protein A and Streptococcus pneumonieae referred
to as protein G. Fc receptors prevents Opsonization by antibodies; binding to the Fc region of
antibody molecules, reversing the orientation of the molecules on the cell.

iv. Surviving within the Phagocyte;

Some bacteria use phagocytosis as an opportunity and do not try to avoid engulfment, this
enables them to hide from antibodies and control some aspects of the immune response,
and they do this by;

a. Escape from the phagosomes: Some bacteria are able to escape from the phagosome
before it fuses with the lysosome (it is when phagosome fuses with lysosome that
destruction occurs) they then multiply within the cytoplasm of the phagocyte protected from
the host defenses. Listeria monocytogenes form pores in the membrane of the phagosome
allowing the bacteria to escape into the cytoplasm.Shigella spp also does the same.

b. Preventing phagosome-lysosome fusion: Salmonella spp are able to sense that they have
been ingested by a macrophage and produce a protein that prevents phagosome-lysosome
fusion thereby avoiding destruction by digestive enzymes.

c. Surviving within the phagolysosome: The fusion of phagosome and lysosome is known a
phagolysosome. Only few organisms can survive the phagolysosome e.g. Coxsiella burnetti
can delay fusion of Phagosome-lysosome to allow it equip itself to survive and grow within
the phagolysosome.

v. Avoiding Antibodies

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 This is one of the major ways; pathogens that survive the innate defenses must find a way of
avoiding the adaptive defenses of which antibodies are the most potent. They do these by;

i. Immunoglobulin A protease: this enzyme cleaves IgA found in the mucous and other
secretions e.g.Neisseria gonorrheae

ii. Antigenic Variation: some pathogens routinely alter the structure of their surface antigens
staying ahead of antibody production. Neisseria gonorrheae is able to vary the antigenic
structure of its pili mimicking host molecules. Pathogens can cover themselves with
molecules that resemble host molecules to appear as “self” to the body e.g. Streptococcus
Pyogenes have capsule composed of hyaluronic acid found in tissues.

4. Damage to the Host:

Damage to the host can be a result of the direct effect of the pathogen, toxins produced or
indirect effect through the immune response.

Toxins:

There are two major types of toxins; Exotoxins and Endotoxins

i. Exotoxins

Exotoxins are proteins produced by both Gram positive and Gram negative bacteria that may
or may not be secreted. Exotoxins are very potent and the major cause of damage to an
infected host. They can be produce by pathogens that has colonized the body surface or
tissue e.g. Corynebacterium diphtherieae or bacterial cells that multiply in a food product
producing the toxins which is then ingested with the food e.g Clostridium botulinum. Most
exotoxins are heat labile (only one known that is heat stable). Because they are proteins, the
body can produce antibodies against them however, many exotoxins are able to cause fatal
damage before adequate immune response is produced hence the need for vaccination
using a toxoid such as Tetanus toxoid and botulinum antitoxin. Most exotoxins fall into three
general mechanism of action;

a. A-B Toxins: the A-B toxins consists of two parts

A- The active subunit

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B- Binding subunit

The A-component is the enzymatic (toxic component) while B-component determines the
cell type the toxin affects by binding to specific receptors on the cell

b. Membrane damaging toxins: These toxins disrupt plasma membrane causing lysing of the
cell; they are called haemolysins because they lyse red blood cells and also cytolysins due to
their ability to lyse other cells.

c. The super antigens: super antigens overrides the specificity of the T-cell response thereby
causing massive release of cytokines by a large number of effector T-cells; normally, an
antigen will stimulate 1 in 10,000 T-cells, a super antigen will stimulate 1 in 5000 T-cells. A lot
of cytokines can be produce the same time, they can also be grouped into functional
categories according to tissues they infect

 Neuro-toxins: Damage nervous system causing symptoms such as paralysis

 Entero-toxins: Damage and cause symptoms associated to intestinal disturbances leading to

diarrhea and vomiting.

 Cytotoxins: Damage different cell types either by interfering with essential cellular
mechanisms or by lysing the cells

ii. Endotoxins:

Endotoxins are lipopolysaccharides which are components of the gram negative cell wall.
Endotoxins cannot be converted to an infective toxoid for immunization. The
lipopolysaccharides are composed of two important parts; Lipid A and O-specific
polysaccharide side chain.

The lipid-A component is responsible for the toxic properties of lipopolysaccharide. The
symptoms associated with endotoxins are due to vigorous innate immune response which
can eliminate the bacteria in a localized infection but become very dangerous in a systemic
infection such as Septicemia.

Endotoxins are heat stable and cannot be destroyed by autoclaving therefore; solutions to
the use of intravenous administration must not only be sterile but must also be free of

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 endotoxins.

Limulus Amoebocyte Lysate (LAL) assay is a very sensitive test used to test the presence of
endotoxins in fluids, the test employs proteins obtained from the blood of horse shoe crab
Limulus polyphemus this form of gel like clot in the presence of endotoxins as little as 10-20
picogram of endotoxins can be detected.

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 Properties Exotoxins Endotoxins
Bacteria source Both Gram negative and Gram negative Species only
Gram positive species
Location in the bacterium Synthesized in the Component of the outer
cytoplasm, may or may not membrane
be secreted
Chemical nature protein Lipopolysaccharide (Lipid A)
Ability to form a toxoid Generally they form toxoid No they are not able
Heat stability Generally inactivated by Heat stable
heat
Mechanism of action A distinct toxic mechanism Innate immune response; A
for each systematic response leads
to fever, a drastic drop in
blood pressure and
disseminated intravascular
coagulation

Damaging Effect of Immune Response:

The immune response generated to eliminate microbes can also damage the host tissue,
this can occur by;

i. Damage associated with inflammation

The inflammatory response can destroy tissue because phagocytic cells recruited to the area
release some of the enzymes and toxic products they contain e.g. in bacterial meningitis and
also complications of some STDs are due to the damage associated with inflammation for
example;Neisseria gonorheae or Chlamydia trachomatis infection are sent from the cervix to
the fallopian tube, the inflammatory response can lead to scaring that obstructs the tube
resulting to either ectopic pregnancy or infertility.

ii. Damage associated with Antibodies: this can occur by two mechanisms;

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a. Antigen-antibody complexes: Complexes may form during immune response and settle in the
kidney or joints causing destructive inflammation e.g. acute glomerulonephritis that is
caused byStreptococcus pyogenes

b. Cross reactive Antibodies: Some antibodies produced as a response to an infection may

binds to the body’s own tissue promoting an auto- immune response e.g. acute rheumatic
fever which is a complication that follows streptococci sore throat may be due to binding of
the antibodies produced in response toStreptococcus pyogenes to a normal tissue proteins.

SEROLOGY

Serology concerns the use of serum antibodies to detect and measure antibodies to detect
and measure antigens or conversely the use of antigens to detect serum antibodies.

The antigen-antibody reaction is a reversible reaction, this reaction is represented as

Antigen + Antibody Antigen-Antibody complex

The forces joining the antigen-antibody complex are not strong covalent bond generally
known as weak interactions. The weak bonds include;

- The Vander waals bond

- Hydrogen bond

- Hydrophobic bond

- Ion-dipole bond

The specific binding between the antigenic determinant known as the epitope and the
antigen combining sites on the immunoglobulin molecule known as the paratope that involve
very small portion of the molecules comprising just a few amino acids and a surface area
between 0.4-8nm2 Specific binding must overcome an overall repulsion between the two
moleculs, multiple binding between the antigen and antibody ensures that the antigen will
bind tightly to the antibody.

The Law of Mass Action

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 At the beginning, a chemical reaction proceed predominantly in one direction but the reverse
rate progressively increases until the forward and the reverse peak equal each other at which
point the reaction is said to have reach equilibrium.

According to the law of mass action, the ratio between the concentration of the product
[complex] and the reactants [Antigen] and [Antibody] is constant at equilibrium. This can be
represented by

[complex]/[Antigen]X[Antibody] = Ka/Kb = Keq

Keq = K- Equilibrium

Keq is called the equilibrium constant and equals to the ratio between the associate (Ka) and
the dissociation (Kb) rate constant. In order to improve antibody detection, the ratio between
the bond and the free antigen i.e. [complex]/[Antigen] should be increased as much as
possible.

Substituting in the formula;

[complex]/[Antigen] = Keq X [Antibody]

From these equation, improving antibody detection can be carried out in two was;

- By increasing the equilibrium constant

- Or by increasing antibody concentration

The problem is that polyclonal antibodies are mixture of antibodies with different affinities so
that the single value for Keq is just an average and does not convey the extent of
heterogeneity.

Antigens and antibodies has some specific terms used in their qualification

I. Specificity: One characteristic of antibodies is that they are specific. Specificity refers to the
ability of an individual antibody combining site to react with only one antigenic determinant

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 or the ability of a population of antibody molecules to react with only one antigen. In general,
there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish
differences in the primary structure of an antigen; Isomeric forms of an antigen as well the
secondary and tertiary structure of an antigen. However, it is not in all cases that antigen-
antibody reaction is specific; sometimes an antibody is to react with more than one antigen
and this is known as cross reactivity

II. Cross Reactivity: Cross reactivity refers to the ability of an individual antibody combining site
to react with more than one antigenic determinant or the ability of a population of antibody
molecules to react with more than one antigen. This arise because the cross reacting antigen
share an epitope in common with the immunizing antigen or because it has an epitope which
is structurally similar to one on the immunizing antigen; this is referred to as multi specificity.

III. Affinity: Antibody affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on the antibody. It is the sum of the attractive and
repulsive forces operating between the antigenic determinant and the combining site of the
antibody. Affinity is the equilibrium constant that describes the antigen-antibody reaction.
Most antibodies have a high affinity for their antigens

IV. Avidity: Avidity is the measure of the overall strength of the binding of an antigen with many
antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence
of the antibody and the valence of the antigen

V. Affinity: refers to the strength of binding between a single antigenic determinant and an
individual antibody combining site where as avidity refers to the overall strength of binding
between multivalent antigens and antibodies.

Immunological Assays

1. Agglutination

Agglutination is the visible clumping together of bacterial cells or particles by an antigen

combining with its specific antibody. The resulting clumps are referred to as agglutinates
while the antibodies involved in agglutination are called agglutinins.

Immunoglobulin- M is a good agglutinin because of its high valency. Agglutination test can

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 be classified as either direct or indirect

a. Direct agglutination; Is used for the detection of antibodies against relatively large cellular
antigen such as those on red blood cells, bacteria and fungi.

b. Indirect or passive agglutination; is used when antibodies against soluble antigens can be
detected by agglutination test. In such cases, the soluble antigens or antibodies are
adsorbed onto inert particles such as latex or carbon particles depending on what has been
adsorbed, indirect agglutination test can be used in detecting specific antibodies to the
soluble antigen or antigen to specific antibody.

Latex agglutination test are now commonly used for the rapid detection of serum antibodies
against many bacterial and viral diseases. Agglutination tests can also be a qualitative or
quantitative test.

In qualitative test, concern is on the presence of the antibody while in quantitative test
concern is on the amount of the antibodies present. In qualitative agglutination test, the
problem encountered is the prozone effect. The prozone reaction or phenomenon is thought
to be as a result of;

- High level of Immunoglobulin-A ( also known as blocking antibodies)

- Nonspecific inhibitory factors

- And excess antibodies

Haemagglutination is used when agglutination reaction involves the clumping of red blood
cells, certain viruses; myxoviruses or paramyxoviridae has the ability to agglutinate red blood
cells without an antigen-antibody reaction, this is called viral haemagglutination.

2. Precipitation Reaction

Precipitation reaction involves the reaction of soluble antigens with antibodies (usually IgM
or IgG) to form visible precipitates. Precipitation usually requires the presence of electrolytes,
carried out in with positive and negative controls.

Precipitation reaction occurs in two stages;

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 First, the antigen and antibody rapidly form antigen-antibody complexes this takes place
within seconds. Next, the antigen-antibody complexes form lattices that precipitate from
solution, this is a slower reaction and may takes minutes to hours.

Precipitation reaction normally occurs only when the ratio of Ag:Ab is optimal (102:103), this
is achieved when separate solution of antigen and antibodies are placed adjacent to each
other and allowed to diffuse together; a cloudy line of precipitation normally called “the ring”
appears in the area in which the optimal ratio has been reached. This area is called the zone
of equivalence.

3. Immunodiffusion

− This is a form of precipitation reaction where the antigen and/or antibody diffused through
an agar or gel agarose medium

− when only the antigen or antibody diffuse through the agar with the corresponding antigen
or antibody being contained in the agar, it is referred to as single diffusion.

− When both the antigen and antibody diffuse, it is called double diffusion.

− In single diffusion, the antigen is incorporated in the agar while in double diffusion, plane
agar is used.

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4. Immunoelectrophoresis
In some tests, electrophoresis is used to speed up the movement of antigen and/or
antibodies in the gel. This technique which combines immunodiffusion and electrophoresis
is known as immunoelectrophoresis. It requires less time; sometimes less than an hour.
Immunoelectrophoresis is the basis of many diagnosis tests including Western blot
(confirmatory test for HIV).

5. Neutralization Reaction

When viruses infect cells, they cause cytopathic effect (CPE) on the cells. Neutralization
reaction involves the inhibition of infection and therefore off cytopathological effect by the
antibody in cell culture or embryonated eggs. If a serum contains antibodies against a
particular virus, the antibodies will prevent the virus from infecting cells in the cell culture or
eggs and off CPE. Neutralization test is both virus and strain specific but it is relatively
complex and not currently commonly used. The most frequently used neutralization test is
the haemagglutination inhibition test (HA). This test is used for the diagnosis of
haemagglutination virus e.g. Myxoviruses.

If a serum contains antibody against these viruses, the antibody will react with and
neutralized the viruses preventing them from agglutinating red blood cells. It is the same with
neutralization reaction in the cell. Both haemagglutination and haemagglutination inhibition
is carried out using microtitre plates (U-bottom)

Haemagglutination; Haemagglutination inhibition;

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6. Immunofluorescence

This is also known as fluorescence antibody test/technique (FAT). It uses fluorescence dye
known as fluorochromes illuminated by UV-light to show the specific lining of the antigen
with its antibody. FAT can be used to identify antigen or to detect the presence of specific
antibody in serum. Fluorescence dyes are combined with antibody to make them
fluorescence when exposed to UV-light. The test is quick, sensitive and very specific but it
requires fluorescence microscope.

FAT can be direct or indiresct;

- Direct FAT: this is used to identify antigens (microbes) in specimen. The specimen is fixed
unto a slide; fluorescein, labeled antibodies are added and the slide is incubated briefly to
allow antigen- antibody reaction. The slide is washed and examined under fluorescence
microscope.

The dye; Fluorescein isothiocynate (FITC) and gives yellow-green florescent.

Direct FAT is used in the identification of many bacteria including Haemophilus influenza,
Neisseria meningitidis, Streptococcus pneumonieae and Clamydia trachomatis. They are
also used for many viruses particularly rabies virus. The fluorochromes acts directly on the
antibody hence direct FAT.

- Indirect FAT: unlabeled antibodies combine with the antigen and the antigen-antibody
complex is dependent on a fluorescent labeled antispecies globulin which attach to the
antibody. An antispecies globulin recognizes all antibodies from the species. If the antibody

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 is of human origin, fluorescent labeled antihuman globulin is used and will react with any
antibody of human origin.

Indirect FAT can be used for detection of antigen and antibody.

- Indirect FAT for Detection of Antibody: Here, a known antigen is placed in the slide and the
serum is added, the preparation is washed and fluorescent labeled antispecies globulin is
added to check for antigen- antibody reaction, there will be fluorescence if there is antigen-
antibody reaction. This is used for diagnosis of syphilis.

- Indirect FAT for Detection of Antigen: A preparation of the specimen is made one a slide and
unlabeled specific antibody is added, the preparation is washed after incubation to remove
excess antibodies. A fluorescence antispecies globulin is added and allowed to combine with
the antibody. The slide is washed again to remove unbound antispecies globulin and then
viewed under fluorescent microscope. The indirect FAT is more sensitive than the direct FAT
and it is more preferred.

7. Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA uses an enzyme system to show the specific combination of an antigen with its
antibody. The antigen or antibody is coated on a solid surface such as a microtitre plate or
plastic test tube or latex articles. There are two main types of ELISA techniques; direct and
indirect ELISA.

i. The Direct ELISA

Direct ELISA is used for the detection of antigens. In direct ELISA, a specific antibody is
coated on the surface of wells of a microtitre plate and the specimen containing the antigen
is added after a period of incubation. The well is washed leaving the antigen- antibody
complexes i. e. if antigen is present. Enzyme labeled specific antibodies often the same as
the one used for coating is then added. The labeled antibody combines with the antigen and
excess unbound antibody is washed off. The antigen is sandwiched between the two
antibodies. A substrate is added and the enzyme acts on the substrate to produce a color
change in the fluid. The enzyme activity is stopped by changing the pH of the reaction or by
denaturing the enzyme. The substrate used must be stable and soluble and must not be
present in any amount in the specimen. The commonly used enzymes are the horseradich
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 peroxidase or alkaline phosphatase. Direct ELISA is used mostly for diagnosis of Rotaviruses.

ii. Indirect ELISA

Here, known antigen is used in coating the inside of the well; the patient serum is added and
incubated for antigen-antibody reaction. The well is washed and enzyme labeled antispecie
globulin is added to react with the antibody. The excess enzyme is washed off and a
substrate added. The presence and concentration of antibody is determined by the
development of color whose intensity will depend on the amount of antibody present.
Indirect ELISA is used in the diagnosis and detection of many bacteria, fungi and viral
infections. When it is used qualitatively to show the presence or absence of antibody, the
result can be read microscopically/ visually. However, when it is used quantitatively a
spectrophotometer is used in reading the result.

MOCROBIOLOGICAL DIAGNOSIS

In diagnosis, the first thing is to ensure that the right specimen is isolated; some of the
samples may be contaminated with normally flora if not properly collected example, a patient
that passes urine. In collection of urine, mid-stream urine (msu) is collected same apply to
sputum. You wouldn’t get the expected result if the right sample is not collected. Samples
should be collected with sterile leak-proof and dry containers. When collecting stool sample,
sterility is not important but for other samples, sterility is very important. The sample should
be accompanied by a request form; after collection. If there is no request form (Which
contains details of the patient) sample will be discarded. If it is a private laboratory, the
request form is opened which contains; the patient name, age and lab Number. The lab
number is different from the hospital number because it is not all patients that comes to the
lab are been allocated hospital number. The form should have the name of the hospital or lab
number, the type of specimen, the date and time of collection which is critical for such
samples e.g. semen analysis; must have time of collection and finally the test required
should be written and noted and sometimes there should be a note on the illness (Doctor’s
diagnosis) which will help in examining the sample

Some Specific Individual Samples;

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1. Sputum

What to looked out for in sputum includes; Mycobacterium tuberculosis, Streptococcus

pneumonieae, Klebsiella pneumonieae and Haemophilus influenza. There are other possible
pathogens such as; Staphylococcus aureus, Yersinia pestis, Pseudomonas auroginosa,
Proteus spp and Streptococcus pyogenes. In laboratory examination of sputum, the first
thing is to examine the appearance of the sputum called macroscopic examination. Sputum
can be purulent, which is mostly green looking and contains mostly pus. A sputum sample
can also be muco-purulent which is green looking with pus but also contains mucus. A
sputum sample can also be mucoid containing mostly mucus and towards whitish in color.
Muco-salivary sputum contain mostly mucus but with a small amount of saliva. If the sample
contains predominantly saliva, normally, that sample is not processed especially when
Mycobacterium tuberculosis is suspected, and the patient is advised to collect deep sputum.

Microscopic Examination of the sputum sample is followed by preparing a smear and stains
such as gram staining is done when looking out for Staphylococcus aureus, S. pneumonieae,
if M. tuberculosis is suspected Ziehl Neelson stain (acid fast stain). You may also do giemsa
preparation if you are looking out for Yersinia pestis and sometimes the respiratory infection
is caused by fungi and not bacteria such asHistoplasma capsulatum

After doing this, you culture the sample. The media of choice for culture sputum is Blood
agar and chocolate agar; if you are sure it is not a serious case, you can use a less
demanding agar such as MacConkey agar. If S. aureus, Klebsiella or Proteus is suspected,
sometimes nutrient agar is used which is not idea. The culture is then incubated at the right
condition and normally overnight or 24hrs incubation. You then examine and read the culture
result

For diagnosis, you set up a sensitivity test after reading the culture. You take the cultures you
have isolated and spread on a Mueller Hilton media (MHA) which is the recommended
medium for all sensitivity tests but in most labs, nutrient media is used. The inoculum is
spread evenly on the surface of the prepared agar by moving the inoculating loop over the
surface. Pick a sensitivity disc such as a multi sensitivity disc ( which have up to 8 antibiotics
in one disc and put it on the surface of the agar) or a single sensitivity disc with one antibiotic
per disc is picked and placed evenly on the plate. This is to enable and observed the zone of

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 inhibition. Zone of inhibition could be reported as; +, ++,+++ or ++++ depending on the extent
of the inhibition.

In most laboratories, diagnosis of M. tuberculosis is stoped at Ziehl Neelson staining and

microscopy because culture of M. tuberculosis requires special reference laboratories. In
control labs, Lowenstein Jensen culture medium is used to isolateM. tuberculosis.

2. Swab

Throat swab, nasal swab, eye swab, ear swab, skin and wound swabs are the items of
referral here. Media of interest in culture of swabs are; MacConkey or Blood agar. After
culture, the microscopic examination is done by staining using gram staining. After
incubation at optimal temperature and time, a sensitivity test is performed.

3. Urogenital specimens

The urogenital specimens include; urethra swabs and normally rectal swabs collected from
males, also high vaginal swabs (HVS) from females. Possible pathogens includes; Neisseria
gonorrheae, Clamydia trachomatis and sometime Trichomonas vaginalis ( may also be seen
in males but not very common), for HVS, we also look out for Candida spp and Gardnerella
vaginalis . Some other pathogens are Streptococcus pyogenes, Staphylococcus aureus,
Herpes simplex viruses (especially in females). If there are genital ulcers, scrappings or
discharge from the ulcers may be collected or the fluid flowing from the ulcers may be
collected. In this case, suspicious pathogens are primarily Treponema pallidum. The rate of
these organisms in urogenital samples is at increase especially in developing countries. The
media of choice for isolation of N. gonorrrhoreae is chocolate agar, blood agar and
macConkey agar may also be used. Sometimes, modified Newyork City (MNYC) medium is
used in standard laboratories. MNYC is a selective media for the isolation ofN. gonorrhoeae .

Microscopic examination: this includes gram staining which is looked out for intracellular
gram negative diploocci (e.g. N. gonorrhoeae ). Others are gram positive in bunches or cocci.

Saline (wet) preparation: two or more drops of saline are dropped into a swab stick and
shake properly. A drop of the saline is then placed onto a slide and the swab used to smear it
on the slide and covered with cover slip.

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 Wet preparation is a special and important test when Candida spp are suspected in women.
Also, wet preparation can be used to report pus cells, epithelial cells and to look out for the
type and amount of epithelial cells. A Geimsa stain can be carried out if Chlamydia
trachomatis is suspected. A dark field examination may also be carried out when looking at
Treponema pallidum .

Sensitivity test: after incubation for 24 hours, a sensitivity test is carried and patients are
asked to come back after 2 days (48 hours) for their results.

In microscopy, yeast cells are more clearly seen under X40 objective. Most of the organisms
that grow on HVS cultures areCandida spp .

4. Faeces or stool specimen

Mostly, what is looked out for in stool specimen are parasites including eggs and cyst (this is
done microscopically) as well as larvae of Strongyloides stercoralis. Possible bacteria
pathogens in stool samples include Shigella spp, Salmonella spp, Campylobacter spp, Vibrio
spp, the pathogenic strains of Escherichia coli (Enteropathogenic E.coli –EPEC,
Enterotoxigenic E.coli -ETEC, Enteroinvasive E.coli –EIEC, Enterohaemorrhagic E.coli –EHEC).
Other pathogens are Clostridium perferinges, Clostridium deficile, Bacillus cereus, S.aureus,
Yersinia enterocolitica. In collection of stool sample, a clean dried disinfectant free vessel
(but not necessary sterile containers are used). Most of the times, the pathogens looked out
for in stool samples in the lab areShigella spp, Salmonella spp andVibrio spp.

NOTE: Campylobacter spp is not very commonly found. Also pathogenic E.coli is not easily
differentiated from the normal E.coli found in stool. Hence, E.coli is exempted from what to
look out for.

Macroscopic examination: colour, consistency (formed, semi-formed or watery). A normal

adult stool will be brown, yellow, black or even brown in colour. Look out for presence of pus
(mucus) or blood stain in the stool. For infant stool, it is normally yellow-green and mostly
semi-formed

Microscopic examination: this is normally by saline or Eosine preparation. Look out for eggs,
cysts or larvae of parasite.

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 Culture: media of choice should be selective such as Salmonella shigella agar (SSA) or
Xylose Lysine Deoxycholate(XLD) agar. XLD is advantageous over SSA because XLD does
not require pre-enrichment with selenite-F broth. Shigella spp will look colourless on SSA,
Salmonella spp will also look colourless on SSA but will have a black colouration at the
centre of the colourless area. On XLD, Salmonella spp and Shigella spp will have red colonies
with Salmonella having black colouraion at its centre. For Vibrio spp, it is grown on TCB and
grows in yellow colonies. After culture, incubation is done at ideal temperature and time
followed by a sensitivity test.

MIU (Motility Indole Urease) is a test that helps to distinguish Salmonella spp and Shigella
spp . Salmonella is motile while Shigella is not motile. Salmonella is indole positive while
Shigella is indole negative. BothSalmonella andShigella are urease negative.

5. Urine specimen

Possible pathogens in urine samples include Streptococcus spp, S.aureus, E.coli, Proteus
spp, Pseudomonas aeruginosa and Klebsiella spp. Other possible pathogens include
Salmonella especially Salmonella typhi and Salmonella paratyphi. Sometimes in heavy
infection, N.gonorrhoea can be found. In some cases, Schistosoma haematobium may be
found

Macroscopic examination: fresh urine (without centrifuging) is put on a slide covered with
cover slip and viewed microscopically. For urine, look out for blood cells which could be due
to acute glomerulonephritis. It can also be an indication of sickle cell diseases. Also, look out
for yeast cells which could be due to vaginal candidiasis. Epithelial cells may also be present
in urine samples in a small amount but when epithelial cells are in large amount, it may be
indication of heavy infection. In urine samples, look out for casts such as hyaline casts, waxy
casts which is an indication of renal damage, cellular casts which resemble hyaline casts but
contain lots of cells, they are indication of inflammation of the pelvis. Granular casts (also
resembles hyaline and cellular casts except that it contains granules) which is also an
indication of renal damage. In addition to casts, crystals may also be found in urine.

i. Cystine crystals: are normally found in cases of congenital disorders.

ii. Tyrosine crystals: is an indication of severe liver damage or liver disease.

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iii. Cholesterol crystals: is an indication of severe kidney disease.

iv. Triple phosphate crystals: are present in alkaline urine. They have no clinical implication.

v. Sulphonamide crystals: they are thicker than tyrosine crystals. They are present when the
patient is undergoing treatment with sulphonamide. They can lead to haematuria (presence
of blood in urine).

vi. Calcium Oxalate crystals: this is normally an indication of calculi (presence of kidney stone).

Parasites commonly found in urine are S.haematobium, T.vaginalis. the urine can be
centrifuged and the pellets of the sediments are gram stained and read.

Culture: media of choice are macConkey agar, blood agar and sometimes nutrient agar. Any
growth below <104 cfu (colony forming units) is not a urinary tract infection (UTI). It must be
5
 10 cfu/ml before it can be treated as a UTI. Estimation of colonies is done by inoculating
and incubating the different concentration of the urine samples and then counting.

Urine chemistry or urinalysis: this test is done using a combi-9 test strip. The strip is dipped
in the urine and colour changes are observed. These tests for the presence of glucose,
protein, biluribin, blood, ketones, pH, urobilinogen e.t.c.

6. Cerebrospinal fluid

Possible common pathogens include Streptococcus.pneumoniae, Neisseriae meningitidis,

Haemophilus influenzae. Others include Staphylococcus aureus, Streptococcus agalacteae
( a group B Streptococcus ), Escherichia .coli, Proteus spp, Pseudomonas aeruginosa,
Salmonella spp. However, interest is usually on Neisseria meningitides. The CSF should be
examined immediately after collection of sample. This is because Neisseriae meningitids is
very fragile and the CSF is normally free of particles and the organism is not very well
protected. CSF samples are normally collected in duplicates (i.e. 2 samples of the CSF).
Sample 1 is normally used for culture and sample 2 is used for other tests. The main reason
for collection of 2 samples is that during needle insertion into the spine, there may be
haemorrhage and the first sample might be blood stained while the second sample is clear.

Macroscopic examination: the CSF sample may be clear, cloudy or definitely purulent. If it is
purulent, a smear is prepared and gram stained because in this case, Neisseriae meningitids
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 is suspected.

Cell count: this is done using a counting chamber. There are two main types of counting
chambers used for CSF; Fuchs Rosenthal ruled chamber and Improved Neubauer chamber

Normal CSF should have about 5 X 106 cells/litre. When the cell count exceeds 5 X 106
cells/litre, it is an indication of meningitis.

Chemistry (chemical tests): the major interest here is presence of glucose and protein.

Culture: chocolate agar is the medium of preference. Blood agar or macConkey agar can also
be used.

Microscopic examination: make a smear, gram stain and look out for intracellular gram
negative diplococcic.

Sensitivity test: after culturing, the grown colonies are used to carry out sensitivity test.

7. Semen

i. Measure the volume of the semen. Normal semen should be within the range of 1.5 – 5.0
mililitre. less than 1.5 is not an ideal volume. Semen analysis should be done after a while of
abstinence.

ii. Estimate the percentage motility of the semen (active, sluggish or dead).

iii. Perform a semen count. Normal semen count ranges between  50 million cells/ml. counting
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 is done by diluting the sample 1 in 20 dilution just like white blood cells (WBC).

iv. Look out for normal shape by staining the semen cells to examine the shape. Nucleus is
stained using Eosin stain and viewed under the microscope.

v. Motility requires that the time of sample collection be noted to effective determination of
motility.

8 Blood culture: there are two types of reaction for blood culture.

i. Tryptone Soya diphasic medium

ii. Thioglycollate broth medium.

The blood sample is collected and immediately inoculated on the media. If Trptone Soya
diphasic medium is used, the plate is examined after 24 hours, and for the following
subsequent five days. After that, the medium is examined once a week for 4 consecutive
weeks. For the Thioglycollate broth medium, the plate is examined after the first 24 hours,
daily for 2 weeks.

BACTERIAL AND PROTOZOAN DISEASES

Bacterial diseases

We are going to be based on tropism i.e. the part of the body that is affected.

1. Skin

Bacterial infection of the skin includes hair follicle; Staphylococcal scalded skin syndrome
(SSSS), Rocky Mountain spotted fever, streptococcal impetigo and lyme disease. Infection of
the hair follicle results in folliculitis; furuncle and carbuncle. Folliculitis is the presence of red
bump or pimple that developed at the site of the involved hair follicle. A furuncle is when the
infection extends from the follicle to adjacent tissues causing localized redness, swelling
tenderness and pains known as furuncle or boil. Pus may drain from the boil. A carbuncle is a
large area of redness swelling and pain with several sites of draining boil. Infection of the
hair follicle is caused mostly by Staphylococcus aureus. S.aureus inhabits the nostrils of
virtually everyone at one point and is normally disseminated to other parts of the body, infect
will occur at an open follicle.
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 Streptococcus impetigo is an infection result from scratches and other injuries that introduce
bacteria into the deeper layers of the epidermis caused mostly by Streptococcus pyogenes
but can also be caused by Staphylococcus aureus. Infection is limited to the epidermis but
bacteria products are absorbed into the circulation. Acute glomerulonephritis is a serious
complication of impetigo caused byStreptococcus pyogenes.

Rocky Mountain spotted fever: so called because it was discovered in the mountain area of
the United States. It is a zoonotic infection maintain by the nature of various species of ticks
and mammals. The disease generally begins suddenly with a headache, pain in the muscles,
joints and fever. A rash develops within a few days that spread throughout the body and then
become raised and hemorrhagic. Bleeding may occur at other site including nose and mouth.
Involvement of the heart, kidney and other tissues results in shock and death except
treatment is given promptly. Rocky Mountain spotted fever is caused byRickettsia rickettsia.

2. The Respiratory System

This is divided into the upper and lower respiratory. The initial refers to the head and the neck
while the later include the chest and the lungs. The upper respiratory infection includes sour
throat, diphtheria and sinus infection. Many of the bacteria that infect the upper respiratory
system such as Haemophilus influenza and β-haemolytic streptococci causes sour throat
but do not required treatment for the immune system can mop them away. The sour throat
caused by Streptococcus pyogenes can involve the pharynx and result in streptococcal
pharyngitis also known as strep throat; one of the major reasons for visit to the hospital in
developed countries. The lymph nodes in the neck are enlarged and tender with abdominal
pain or headache.

Diphtheria is a deadly toxin mediated disease that usually starts as a mild sore throat slit
fever but eventually leads to heart and kidney failure and paralysis. Diphtheria has drastically
reduced worldwide as a result of childhood immunization. Diphtheria is caused by
Corynebacterium diphtherieae.

3. The Lower Respiratory System:

Diseases include Pneumonias, whooping cough (pertussis), Tuberculosis and Legionnaires

disease.

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 Pneumonia involves the accumulation of fluid in the alveoli of the lungs. This is usually
coughed out as sputum; preceded by running nose; shortness of breath resulting from the
fluid accumulation in aveoli and patient can die for lack of oxygen. The major causative agent
of pneumonia is Streptococcus pneumoniae but can also be caused by Klebsiella
pneumoniae and Mycoplasm pneumoniae.

Pneumococcal pneumonia complication includes; septicemia, endocarditic and meningitis,

which occurs when bacteria enters the blood stream.

Tuberculosis: is a chronic disease characterized by slit fever, progressive weight loss,

sweating at night and productive blood streaked sputum. Tuberculosis has reduced
drastically especially in industrialized countries due to improved living standard but has been
compounded by the HIV/AIDS epidemic and drug resistance which can result to multidrug
resistance; tuberculosis and extensive drug resistant tuberculosis.

4. The Alimentary Canal (GIT) is also divided into upper and lower GIT. The upper goes from the
mouth down to the stomach while the lower system includes the intestine, liver and the
pancreas. The disease of the upper alimentary tract includes; Dental carries (tooth decay),
periodontal disease, trench mouth and helicobacter pylori gastritis.

Helicobacter pylori gastritis usually leads to peptic ulcer characterized by localized

abdominal pain, tenderness and bleeding. It affects the stomach and upper part of the
duodenum. The bacterial infection of the lower GIT includes; Cholera, Salmonellosis and
Campylobacteriosis, E. coli gastroenteritis; all this are forms of gastroenteritis with some
peculiarities.

Cholera is severe watery diarrhoea that can lead to loss of 20litres of fluid per day.

Vomiting occurs in most people at the onset of the disease, the main symptoms includes;
dysentery, fever, headache, joint pains, stiff neck, convulsion may occur.

The most severe form of salmonellosis caused bySalmonella typhi. In typhoid, the organism
escape the host immune system and it is carried by the blood stream all over the body
resulting in prolonged fever, absecess, septicemia and shock with little or no diarrhea. The
major problem of gastroenteritis is normally dehydration resulting from loss of fluid.

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5. Genitourinary infection

This can also be divided into urinary and genital infection. The genital infection is further
divided into venereal and non venereal disease. The disease of the UTI includes bacteria
cystitis, urethra infection, kidney and leptospirosis. The most common of the UTI is cystitis
(inflammation of the bladder) many bacterial species can cause UTI but the most common is
E. coli.

Leptospirosis is a zoonotic infection where the urinary tract is infected from the blood
stream. Symptoms includes; prompt onset of headache, spiking fever, chills and severe
muscle pains usually caused byLeptospira interogans.

The non venereal bacterial diseases are the bacterial vaginosis and staphylococcal toxic
shock.

Bacterial vaginosis is caused by many bacteria but the most common is Gardnerella
vaginalis. The venereal bacteria disease includes gonorrhea, syphilis and chlamydia infection
Cancroids.

Gonorrhea in men is characterized by urethritis with pains during urination on a pick pus
containing discharges from the penis. The symptoms usually lead to prompt treatment. In
women, they usual symptoms are; painful urination and vaginal discharge is mild and usually
overlooked. Complications from non-treatment infection in men can lead to partial
destruction of the urethra that can spread to the prostate gland and testis resulting in
Orchitis (Inflammation of the testis) and sterility.

In women, complications include progression of infection through the uterus down the
fallopian tube causing pelvic inflammatory diseases resulting into sterility or ectopic
pregnancy. The eyes of the new born baby can be infected from a mother with symptomatic
or asymptomatic gonorrhea leading to blindness this is known as Ophthalmia neonatorum
which can be prevented by droping 1% of silver nitrate or any antibiotic infusion in the eye of
the new born infant within one hour of delivery. Gonorrhea is caused byNeisseria gonorrheae.

Clamydia comes with symptoms the same as gonorrhea about 25-40% of those suspected
with gonorrhea has chlamydia, the causative agent isChlamydia trachomatis

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 Syphilis occurs in many forms and passes through three clinical stages

- The primary syphilis; occur three weeks after infection and involves a painless ulcer with a
hard ring called hard chancre. This appears at the site of infection after 2-10 weeks.

- The manifestation of secondary syphilis includes; running nose, watery eyes, aches and
pains, sore throat and rashes on the palm and sores. T his is followed after a latent period
that can last for many years.

- Manifestation of tertiary syphilis results into mental illness, blindness, stroke and other
nervous system disorders.

6. Disease of the Nervous system

The bacterial diseases of the nervous system includes; meningitis, Listerosis, Leprosy
(Hansen’s) and Botulism

Menigitis may be caused by a number of bacteria including Heamophilus influenza,

Streptococcus pneumonieae and Neisseria menigiditis which occurs in children; 6-11 months,
death also occurs frequently in older children and adult. Symptoms of bacterial meningitis
are similar and usually begin with mild cold, followed by sudden onset of severe headache,
fever, pain, stiff neck and back, nausea and vomiting, deafness and changes in
consciousness which may progress to comma and developed to shock and death within
24hrs.

Wound infection may be divided into those caused by aerobic organisms and those caused
by anaerobes. The common wound infections are caused by Staphylococcus spp,
Pseudomonas aroginosa and proteus species. This infection usually delays wound healing.
The more dangerous wound infectious organisms in the aerobic group are those caused by
group A streptococcus.

Vibrio vulnificus called flesh eater can cause infection sometimes resulting in necrotizing
fasciitis (flesh eating). The wound infection by anaerobic bacteria is tetanus and gas
gangrenes, tetanus caused by Clostridium tetani and gas gangrenes caused by Clostridium
perfrengens.

7. Bacterial Disease of Blood and Lymphatic system.

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 The bacterial disease of blood and lymphatic system are those caused by blood infections
notably sub-acute bacteria endocarditis involving the valves of the heart caused mainly by
Staphylococcus epidermidis, other bacterial species can also cause sub-acute bacteria
endocarditis. Those that involves lymph nodes and spleen includes; tularemia infection
(rabbit fever), Brucellosis (undulant fever) and Plague (Black Death).

PROTOZOAN DISEASE OF HUMANS

Most of the protozoan diseases in man are those involving the lower alimentary tract canal,
the blood and the lymphatic system. Those involving the lower alimentary tract include
giardiasis which is caused by Gardia lamblia and symptoms of giardiasis can be mild
including indigestion, gas in the stomach and nausea can also be severe. In severe cases,
symptoms include vomiting, explosive diarrhea, abdominal cramps, fatigue and weight loss.

Another disease is cryptosporidiosis which is caused by Cryptosporidium spp but notably

Cryptosporidium parvum . Cryptosporidiosis is characterized by fever, loss of appetite,
nausea, cramping abdominal pains and profuse watery diarrhea. Cryptosporidiosis became
important following the HIV/AIDS pandemic/epidemic but is known to be the problem of
both the healthy and immune comprised individuals.

Cyclosporiasis: this is caused by Cyclospora cayetanensis. It begins after about 1 week of

incubation with fatigue, loss of appetite, slight fever, vomiting and watery diarrhea which is
normally followed by weight loss. Diarrhea subsides in 3-4 days but relapses for up to 4
weeks. Oocysts of cyclospora unlike those of cryptosporidium are immature when released
with stool so that person to person infection is not possible but person to person infection is
possible in cryptosporidiosis because the oocyst is infectious.

Amobiasis is caused mainly by Entamoeba histolytica . The disease is usually asymptomatic

but can result to chronic mild diarrhea lasting for months or years to acute diarrhea/
dysentery. Protozoan diseases of the blood are malaria, leishmaniasis and trypanosomiasis.
Malaria is caused by Plasmodium spp such as Plasmodium falciparum, Plasmodium vivax,
Plasmodium ovale and Plasmodium malariae. Symptoms of malaria are fever, headache,
pains in the joints and muscles and these symptoms last for 2 – 3 weeks and after, the

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 symptoms changes and tend to fall into 3 phases.

i. The patient abruptly feels cold and develops shaking chills which last for about an hour and
this is the cold phase.

ii. Following the chills, temperature rises steeply often reaching 40oc and this is known as the
hot phase.

iii. After hours of fever, temperature falls and drenching sweat occurs and this is known as the
wet phase. The patient feels well for about 24-48 hours later depending on the species of the
malaria and this process is repeated again.

Protozoan Sexually Transmitted Diseases (STD’s).

The protozoan STD’s include;

i. Trichomoniasis: this is caused by the organism Trichomonas vaginalis . Most of the

trichomonas infections (symptomatic infections) occurs in women and the symptoms
include itching of the vulva and inner thighs, itching and burning of the vagina, yellowish
green discharge with foul smelling odour and at times, burning discomfort with urination.
Most infections in men are asymptomatic but they may be penile discharge (from penis),
burning pain with urination, painful testes or a tender prostate gland.

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