MICROBIOLOGY

CIMS 0121
By Jimmy Ongori
©2012

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 LECTURE ONE

INTRODUCTION AND HISTORY OF

MICROBIOLOGY

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Lectures on;
 Monday 10.00 – 12.00
 Tuesday 4-6pm

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 INTRODUCTION
Microbiology is the study of microorganisms -
organisms which are of microscopic
dimensions.
These organisms are too small to be perceived
by the unaided human eye.
If an object has a diameter of less than 0.1mm,
the eye can not perceive it at all, and very little
detail can be perceived in an object with a
diameter of I mm.
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 INTRODUCTION Cont’
Generally organisms with a diameter of 1 mm or
less are microorganisms and fall into the broad
domain of microbiology.
Since most microorganisms are only a few
thousandths of a millimetres in size they can
only be seen with the aid of microscope
These include protozoa, algae, fungi and
bacteria. Viruses are ultramicroscopic and have
an obligate parasitic relationship, but they still
come under the domain of microbiology.
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 What is Microbiology?
 Micro - too small to be seen with the naked
eye
 Bio - life
 Ology - study of

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 Organisms included in the study of
Microbiology
1. Bacteria  Bacteriology
2. Protozoans  Protozoology
3. Algae  Phycology
4. Parasites  Parasitology
5. Yeasts and Molds
 Fungi  Mycology
6. Viruses  Virology

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EARLY HISTORY OF MICROBIOLOGY
Historians are unsure who made the first
observations of microorganisms, but the
microscope was available during the mid-1600s,
and an English scientist named Robert Hooke
made key observations.
In 1665 he observed strands of fungi among the
specimens of cells he viewed.
He proposed the Cell Theory - all living things
are made up of cells

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 History of Microbiology
In the 1674, Anton van Leeuwenhoek was 1st
person to view microorganisms
Made careful observations of microscopic
organisms, which he called animalcules.
Until his death in 1723, van Leeuwenhoek
revealed the microscopic world to scientists of
the day and is regarded as one of the first to
provide accurate descriptions of protozoa, fungi,
and bacteria.

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 Spontaneous generation
Theory that life just ―spontaneously‖ developed
from non-living matter
After van Leeuwenhoek died, the study of
microbiology did not develop rapidly because
microscopes were rare and the interest in
microorganisms was not high.
In those years, scientists debated the theory of
spontaneous generation, which stated that
microorganisms arise from lifeless matter such
as beef broth.
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History

 In 1668 the theory of spontaneous generation

was disputed by Francesco Redi, who showed
that fly maggots do not arise from decaying
meat (as others believed)
 To prove this he covered the meat with gauze
to prevent entry of flies. Hence they could not
deposit their eggs. No maggots appeared

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Schleiden and Schwann
Formulated Cell Theory: cells are the
fundamental units of life and carry out all the
basic functions of living things
Pasteur, FR and Tyndall, UK (1861)
Finally disproved Spontaneuous Generation.
Joseph Lister, UK (1867)
Used phenol (carbolic acid) to disinfect
wounds
First aseptic technique in surgery

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Robert Koch, (1876)
Postulates – Germ theory (1876)
Identified microbes that caused anthrax
(1876), tuberculosis (1882) and cholera (1883)
Developed microbiological media & streak
plates for pure culture (1881)
Gave rise to Koch’s postulates

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 Koch’s Postulates
1. The specific causative agent must be found in
every case of the disease.
2. The disease organism must be isolated from
the lesions of the infected case and
maintained in pure culture.
3. The pure culture, inoculated into a susceptible
or experimental animal, should produce the
symptoms of the disease.
4. The same bacterium should be re-isolated in
pure culture from the intentionally infected
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animal.
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 Pasteur’s Contribution to Microbiology
 Pasteur showed that microorganisms are not
evenly distributed in the atmosphere and that
their number varies from place to place.

 For this, he took a large number of sealed flasks

containing boiled and cooled infusions and
opened a few at a time for a short period at
various places and resealed.

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Pasteur designed special “swan-necked flasks” with
a boiled meat infusion

Shape of flask
allowed air in
(vital force) but
trapped dust
particles which
may contain
microbes

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Out of the 20 flasks which he opened and
resealed on a dusty road, 8 showed spoilage
Out of the 20 that he opened on the top of a
mountain, only five showed spoilage
Out of 20 that he opened near a glacier, only
one showed spoilage.
During the short time that the flasks were open,
air had rushed into the flasks carrying along with
it the microorganisms. After resealing and
incubation, only those flasks which got the
microbes from the air showed growth and
spoilage.
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From these experiments he concluded that the
air contained microbes and the number of
microorganisms in the atmosphere varied from
place to place
He also made an intensive study of the beer
and wine manufacturing processes and causes
of souring and spoilage of beer and wines. He
found that wine spoilage was caused by the
growth of undesirable contaminating microbes
which produced the so called "disease".
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The solution to this problem lay in preventing
the growth of undesirable organisms.
After considerable experimentation Pasteur
showed that wine did not undergo spoilage if it
was held for a few minutes at 50 to 60°C. In the
same way, he found that beer could also escape
the ―disease‖ by heating to 50-55° C.
This gave rise to the new process of preserving
wine, fruit juices, milk etc., and was called
"Pasteurization".

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 The Germ theory of Disease

1835: Agostino Bassi showed a silkworm disease

was caused by a fungus.
1865: Pasteur believed that another silkworm
disease was caused by a protozoan.
1840s: Ignaz Semmelwise advocated hand
washing to prevent transmission of puerperal
fever
1860s: Joseph Lister used a chemical disinfectant
to prevent
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1796: Edward Jenner inoculated a person with
cowpox virus. The person was then protected
from smallpox.
Called vaccination from vacca for cow
The protection is called immunity

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 Chemotherapy – treatment with chemicals

 1910: Paul Ehrlich developed a synthetic

arsenic drug, salvarsan, to treat syphilis.
 1930s: Sulfonamides were synthesized
 1928: Alexander Fleming discovered the first
antibiotic - penicillin, that killed Staphylococcus
aureus.

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DEFINITION OF SOME TERMS
i. Pathogen – disease causing microorganism
ii. Infection – the process of transmission of
disease by a disease causing MO
iii. Pathogenesis – process by which disease
starts and develops within the body

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 Benefits of MOs
 Maintain balance of environment (microbial ecology)
 Basis of food chain
 Nitrogen fixation
 Photosynthesis
 Digestion, synthesis of vitamins
 Manufacture of food and drink, drugs
 Genetic engineering
 Synthesis of chemical products
 Recycling sewage
 Bioremediation: use microbes to remove toxins (oil spills)
 Use of microbes to control crop pests
 Normal microbiota
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 LECTURE TWO

BACTERIAL GROWTH

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 GROWTH
Growth is the orderly increase in the sum of all
the components of an organism.
Increase in size that results when a cell takes
up water or deposits lipid or polysaccharide is
not true growth.
Cell multiplication is a consequence of growth;
in unicellular organisms, growth leads to an
increase in the number of individuals making up
a population or culture.

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 GROWTH Cont’
Microbial concentrations can be measured in
terms of;
 cell concentration (the number of viable cells per
unit volume of culture) or
 biomass concentration (dry weight of cells per
unit volume of culture).
Culture - is a method of propagating/multiplying
microbial (living tissue cells) in media conducive
to their growth

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 GROWTH Cont’
Most Bacteria Reproduce by Binary Fission
The cell doubles in size
Replicates the chromosome (DNA)
Forms a septum in the center
Synthesizes a Cell Wall at the Septum
Daughter cells separate.

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 Binary fission

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 GROWTH Cont’
 All microorganisms undergo similar growth
patterns.
 Each growth Curve has 4 Phases
 Lag phase
 Logarithmic phase
 Stationary phase
 Death phase

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 Microbial Growth Curve

Stationary phase
# cells / ml

Log phase
Death
phase

Lag phase

Time
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 1. LAG PHASE
The lag phase represents a period during which
the cells, depleted of metabolites and enzymes
as the result of the unfavorable conditions that
existed at the end of their previous culture
history, adapt to their new environment.
Enzymes and intermediates are formed and
accumulate until they are present in
concentrations that permit growth to resume.

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 LAG PHASE
If the cells are taken from an entirely different
medium, it often happens that they are
genetically incapable of growth in the new
medium.
In such cases a long lag may occur,
representing the period necessary for a few
mutants in the inoculum to multiply sufficiently
for a net increase in cell number to be apparent.

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 2. LOGARITHMIC/EXPONENTIAL PHASE
During the exponential phase, the cells are in a
steady state.
New cell material is being synthesized at a
constant rate, but the new material is itself
catalytic, and the mass increases in an
exponential manner. This continues until one of
two things happens: either one or more
nutrients in the medium become exhausted, or
toxic metabolic products accumulate and inhibit
growth.
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 2. LOG PHASE
For aerobic organisms, the nutrient that
becomes limiting is usually oxygen.
Population doubles every generation
Microbes are sensitive to adverse conditions
e.g.
 Antibiotics
 Anti-microbial agents

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 3. STATIONARY PHASE
Eventually, the exhaustion of nutrients or the
accumulation of toxic products causes growth to
cease completely.
In most cases, however, cell turnover takes
place in the stationary phase: There is a slow
loss of cells through death, which is just
balanced by the formation of new cells through
growth and division. When this occurs, the total
cell count slowly increases although the viable
count stays constant.
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 3. STATIONARY PHASE
Cells begin to encounter environmental stress
due;
 Lack of nutrients
 Lack of water
 Not enough space
 Metabolic wastes
 Oxygen
 pH

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 4. DEATH PHASE (PHASE OF DECLINE)
After a period of time in the stationary phase, which
varies with the organism and with the culture
conditions, the death rate increases until it reaches
a steady level.
Death due to limiting factors in the environment
In most cases the rate of cell death is much slower
than that of exponential growth.
After the majority of cells have died, the death rate
decreases drastically, so that a small number of
survivors may persist for months or even years.
This persistence may in some cases reflect cell
turnover, a few cells growing at the expense of
nutrients released from cells that.
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 REQUIREMENTS FOR GROWTH

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Growth refers to increase in number of cells
In terms of a single cell it is seen as an increase
in size and mass over time
Growth requirements can be divided into;
1. Physical – temperature, pH, osmotic pressure
2. Chemical – water, micro and macro-nutrients,
organic growth factors

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 TEMPERATURE
It is one of the most important environmental
variables affecting microbial growth
Every MO possesses a characteristic range of
temperatures over which it can grow
For a particular MO there is a minumum,
maximum and optimum temperature
 Minimum – lowest temp’ at which the spp. Can
grow
 Optimum – temp’ at which spp grows best
 Maximum – highest temp’ at which growth is
possible
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 TEMPERATURE
Psychrophilic 0-200 C but grow best at low
temperatures (15–20 °C)
Mesophilic forms grow best at 30–37 °C
Thermophilic forms grow best at 50–60 °C.
Some organisms are hyperthermophilic and can
grow above the temp’ of boiling water, which exists
under high pressure in the depths of the ocean.
Most organisms are mesophilic; 30 °C is optimal
for many free-living forms, and the body
temperature of the host is optimal for symbionts of
warm-blooded animals.
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 Psychrophiles
Some can grow at 00C, but will not grow beyond
200 C
Some, optimum is 20-300 C – common in food
spoilage because can grow at refrigerator temp’

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 pH
Every organism has a range of pH over which
growth is possible and an optimal pH
Most bacteria (neutralophiles) grow between
pH 6.5 - pH 7.5
Acidophilic bacteria – grow in acidic pH eg
Lactobacillus which produce acid. Most fungi
grow at 1.5-2.0
Alkalophiles – can grow upto pH10.5

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 OSMOTIC PRESSURE
 Hypertonic environments, increase salt or
sugar, cause plasmolysis
 Extreme or obligate halophiles require high
osmotic pressure
 Facultative halophiles tolerate high osmotic
pressure

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 The Requirements for Growth:
Physical Requirements

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Figure 6.4
 OXYGEN
Many forms of life require oxygen for aerobic
respiration
Some forms of oxygen can actually be toxic
Metabolism, UV light, chemical reactions can
create toxic forms of oxygen
Aerobes have enzymes to detoxify toxic forms
of oxygen

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 Toxic Forms of Oxygen
 Singlet oxygen: O boosted to a higher-energy state
 Superoxide free radicals: O2

 Peroxide anion: O22

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 OXYGEN

Obligate aerobes – require O2 to survive

Facultative anaerobes – can grow in the
absence of O2 eg E. coli
Obligate anaerobes – unable to use molecular
O2 eg Clostridia
Microaerophilic – aerobic but grow only in O2
concentrations lower than those in the air

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 Oxygen (O2)
obligate Faultative Obligate Aerotolerant
Microaerophiles
aerobes anaerobes anaerobes anaerobes

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 ASSIGNMENT
Write short notes on the following;
Chemical requirements for microbial growth eg
carbon, nitrogen, sulphur etc
Classify MOs according to requirements eg
autotrophs etc

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 LECTURE 3

CULTURE MEDIA

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Culture is the term given to microorganisms
that are cultivated in the lab for the purpose of
studying them.

Medium is the term given to the combination of

ingredients that will support the growth and
cultivation of microorganisms by providing all
the essential nutrients required for the growth
(that is, multiplication) in order to cultivate these
microorganisms in large numbers to study them.

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Organisms have varying needs hence, culture
media have also been formulated with different
ingredients.
Culture media may be found as;
 liquid (called broth)
 semi-solid
 solid.
Media are solidified by the addition of solidifying
agents such as agar (inert compound).
Varying the concentration of agar will yield
varying degrees of solidification.
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 Culture media may be classified as:
1. Synthetic media (Defined)
2. Complex (Non-synthetic) media
Synthetic media contain only ingredients for
which a complete chemical formula is known.

Complex media contain at least one ingredient

for which a chemical formula is not known
(such as milk, egg, malt, animal tissues)

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Culture media can also be classified based on
the function they perform in determining various
characteristics of organism that are able to grow
on/in them
 e.g. Differential, Selective media.

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The primary function of culture media is to be able
to grow particular organisms on/in them.
Media should be devoid of any other living
organisms.
This is done through the process of sterilization (a
process by which all living organisms and their
spore forms are killed and the medium is made
sterile)
Sterilized through the process of autoclaving
(using high temperatures that will kill all living
organisms under increased pressure for specified
periods of time – in an appliance called the
autoclave)
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 Types of media
For the cultivation of bacteria, a commonly used
medium is nutrient broth, a liquid containing
proteins, salts, and growth enhancers that will
support many bacteria.
To solidify the medium, an agent such as agar
is added. Agar is a polysaccharide that adds no
nutrients to a medium, but merely solidifies it.
The medium that results is nutrient agar.
Very few bacteria can decompose agar

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 COMPLEX MEDIA
Many media for microorganisms are complex,
reflecting the growth requirements of the
microorganisms.
Usually contain complex materials of biological
origin eg milk, blood or yeast extract
For instance, most fungi require extra carbohydrate
and an acidic environment for optimal growth. The
medium employed for these organisms is potato
dextrose agar, also known as.
 For protozoa, liquid media are generally required,
and for rickettsiae and viruses, living tissue cells
must be provided for best cultivation.
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 Selective Media
Inhibits the growth of some bacteria while
selecting for the growth of others eg
1. Brilliant Green Agar
 dyes inhibit the growth of Gram (+) bacteria
 selects for Gram (-) bacteria
 Most G.I.T infections are caused by Gram (-) bact.
2. EMB (Eosin Methylene Blue)
 dyes inhibit Gram (+) bacteria
 selects for Gram (-) bacteria
 G.I. Tract infections caused by Gram (-)
bacteria
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 Differential Media
Differentiates between different organisms
growing on the same plate
Example:
Blood Agar medium (TSA with 5% sheep
blood)
used to differentiate different types of
Streptococci
o Alpha-Hemolysis- incomplete RBC lysis
o Beta Hemolysis- complete RBC lysis
o Gamma Hemolysis- no RBC lysis
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 Selective and Differential Media
Mannitol Salt Agar
 used to identify Staphylococcus aureus
 High salt conc. (7.5%) inhibits most bacteria
 Sugar - Mannitol
 pH Indicator (Turns Yellow when acidic)
MacConkey’s Agar
 used to identify Salmonella
 Bile salts and crystal violet (inhibits Gram (+) bact.)
 Sugar - lactose
 pH Indicator
 Many Gram (-) enteric non-pathogenic bacteria can
ferment lactose, Salmonella can not
jimmy ongori 2013
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 Enrichment Broths
―Encourage‖ the growth of a particular type of
microbe;
Addition of ―nutrients‖ enrich for microbial
group of interest eg
o Cellulose broth- enriches for microbes which
degrade cellulose
o Petroleum Broth- enriches for microbes which
could eat an oil spill.

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Bacterial Morphology Arrangement
1. Bacilli
a.Streptobacilli
b. Bacilli

2. Cocci
a. Cocci
b. Doplococci
c. Streptococci
d. Staphylococci

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 Bacterial Morphology Arrangement
3 Spiral
a. Vibrio
b. Spirillum
c. Spirochete

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 Rod-Shaped Bacteria

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 Spherical Bacteria
Diplococcus
Streptococcus
Staphylococcus

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 Spiral-Shaped Bacteria

Borrelia burgdorferi
Spirochete:

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 METHODS OF STUDY

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 OPTICAL METHODS
1. Light microscope
a. Bright field microscope
b. Phase contrast microscope
c. Dark field microscope
2. Fluorescence Microscope
3. Differential Interference Contrast (DIC)
Microscope
4. The Electron Microscope
5. Confocal Scanning Laser Microscope
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 1. The Light Microscope

The resolving power of the light microscope

under ideal conditions is about half the
wavelength of the light being used. (Resolving
power is the distance that must separate two
point sources of light if they are to be seen as two
distinct images.)
The useful magnification of a microscope is the
magnification that makes visible the smallest
resolvable particles.
Several types of light microscopes are
commonly
72 used in microbiology:
jimmy ongori 2013 03/11/2013 21:18
 a. Bright-Field Microscope
 Is most commonly used in microbiology and consists of two
series of lenses (objective and ocular lens)
 Generally uses a 100-power objective lens with a 10-power
ocular lens, thus magnifying the specimen 1000 times. Particles
0.2 μ in diameter are magnified to about 0.2 mm and so become
clearly visible.
 Specimens are rendered visible because of differences in
contrast between them and the surrounding medium.
 Many bacteria are difficult to see well because of their lack of
contrast with the surrounding medium.
 Dyes (stains) can be used to stain cells or their organelles and
increase their contrast so that they can be more easily seen in
the bright-field microscope.

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 b. Phase Contrast Microscope
Was developed to improve contrast differences between
cells and surrounding medium, making it possible to see
living cells without staining them; with bright-field
microscopes, killed and stained preparations must be used.
Takes advantage of the fact that light waves passing through
transparent objects, such as cells, emerge in different
phases depending on the properties of the materials
through which they pass.
This effect is amplified by a special ring in the objective lens
of a phase contrast microscope, leading to the formation of
a dark image on a light background.

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 c. Dark-Field Microscope
Light microscope in which the lighting system has
been modified to reach the specimen from the sides
only.
This creates a "dark field" that contrasts against the
highlighted edge of the specimens
Resolution by dark-field microscopy is quite high.
Thus, this technique has been particularly useful for
observing organisms such as Treponema pallidum, a
spirochete which is less than 0.2 μm in diameter and
therefore cannot be observed with a bright-field or
phase contrast microscope
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 FLUORESCENCE MICROSCOPE
Used to visualize specimens that fluoresce - the
ability to absorb short wavelengths of light
(ultraviolet) and give off light at a longer wavelength
(visible).
Some organisms fluoresce naturally because of the
presence within the cells of naturally fluorescent
substances such as chlorophyll. Those that do not
naturally fluoresce may be stained with a group of
fluorescent dyes called fluorochromes.

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Widely used in clinical diagnostic microbiology eg
the fluorochrome auramine O, which glows yellow
when exposed to ultraviolet light, is strongly
absorbed by Mycobacterium tuberculosis, When
applied to a specimen containing M tuberculosis and
exposed to ultraviolet light, the bacterium can be
detected by the appearance of bright yellow
organisms against a dark background.

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The principal use of fluorescence microscopy is a
diagnostic technique called the fluorescent-
antibody (FA) technique or
immunofluorescence. In this technique, specific
antibodies (eg, antibodies to Legionella
pneumophila) are chemically labeled with a
fluorochrome
If the specimen contains L pneumophila, the
fluorescent antibodies will bind to antigens on
the surface of the bacterium, causing it to
fluoresce when exposed to ultraviolet light.
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Differential Interference Contrast (DIC) Microscope
 DIC microscopes employ a polarizer to produce polarized light.
The polarized light beam passes through a prism that generates
two distinct beams; these beams pass through the specimen and
enter the objective lens where they are recombined into a single
beam.
 Because of slight differences in refractive index of the
substances each beam passed through, the combined beams are
not totally in phase but instead create an interference effect,
which intensifies subtle differences in cell structure. Structures
such as spores, vacuoles, and granules appear three
dimensional.
 DIC microscopy is particularly useful for observing unstained
cells because of its ability to generate images that reveal internal
cell structures that are less apparent by bright-field techniques.
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 The Electron Microscope
The high resolving power of the electron microscope has enabled
scientists to observe the detailed structures of prokaryotic and
eukaryotic cells.
Two types of electron microscopes in general use: the
transmission electron microscope (TEM), and the scanning
electron microscope (SEM).
The TEM was the first to be developed and employs a beam of
electrons projected from an electron gun and directed or focused
by an electromagnetic condenser lens onto a thin specimen.
TEM can resolve particles 0.001 m apart. Viruses, with diameters
of 0.01–0.2 m, can be easily resolved.
The SEM is useful for providing three-dimensional images of the
surface
80 of microscopic objects.
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 Confocal Scanning Laser Microscope

 A laser beam is bounced off a mirror that directs the beam through a
scanning device. Then the laser beam is directed through a pinhole that
precisely adjusts the plane of focus of the beam to a given vertical layer
within the specimen.
 By precisely illuminating only a single plane of the specimen, in a
relatively thick specimen, various layers can be observed by adjusting the
plane of focus of the laser beam.
 Cells are often stained with fluorescent dyes to make them more visible.
Alternatively, false color images can be generated by adjusting the
microscope in such a way as to make different layers take on different
colors. The CSLM is equipped with computer software to assemble
digital images for subsequent image processing.

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 STERILIZATION AND
DISINFECTION

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 Sterilization and Disinfection
Sterilization is defined as the process where all the living
microorganisms, including bacterial spores are killed.
 Sterilization can be achieved by physical, chemical and
physiochemical means.
Disinfection: Reducing the number of pathogenic
microorganisms to the point where they no longer cause
diseases. Usually involves the removal of vegetative
pathogens
 Chemicals used in disinfection are called disinfectants.
 Not all disinfectants can kill all microorganisms. Some
methods of disinfection such as filtration do not kill
bacteria, they separate them out.
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 Sterilization and Disinfection
Sterilization is an absolute condition while
disinfection is not. The two are not synonymous.
Decontamination is the process of removal of
contaminating pathogenic microorganisms from
articles by a process of sterilization or disinfection.
It is the use of physical or chemical means to
remove, inactivate, or destroy living organisms on a
surface so that the organisms are no longer
infectious.

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 Sterilization and Disinfection
Asepsis: is the employment of techniques (eg gloves, air
filters, uv rays etc) to achieve microbe-free environment.
Antisepsis: is the use of chemicals (antiseptics) to make skin
or mucus membranes free of pathogenic microorganisms.
Bacteriostasis: is a condition where the multiplication of the
bacteria is inhibited without killing them.
Bactericidal: Chemicals that can kill or inactivate bacteria.
Have various names such as bactericidal, virucidal,
fungicidal, microbicidal, sporicidal, tuberculocidal or
germicidal.

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 The Ideal Disinfectant
 Resistant to inactviation
 Broadly active (killing pathogens)
 Not poisonous (or otherwise harmful)
 Penetrating (to pathogens)
 Not damaging to non-living materials
 Stable
 Easy to work with
 Otherwise not unpleasant
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Disinfectant Performance…
 Is dependent on Disinfectant concentrations
 Is dependent on length (time) of administration
 Is dependent on temperature during administration
(usual chemical reaction 2x increase in rate with each
10°C increase in temperature)
 Microbe type (e.g., mycobacteria, spores, and certain
viruses can be very resistant to disinfection—in general
vegetative cells in log phase are easiest to kill)
 Substrate effects (e.g., high organic content interferes
with disinfection—stainless steel bench easier to
disinfect than wood)
 It is easier (and faster) to kill fewer microbes than many
87 microbes
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88 jimmy ongori 2013 03/11/2013 21:18
 PHYSICAL METHODS
1. Sunlight
2. Heat
a) Dry heat – red heat, flaming heat, incineration,
hot air oven, infra red heat
b) Moist heat – at 1000 C, >1000 C, < 1000 C
3. Vibration
4. Radiation – non-ionizing and ionizing
5. Filtration

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 PHYSICAL METHODS OF STERILIZATION
1. Sunlight
o The microbicidal activity of sunlight is mainly due
to the presence of ultra violet rays in it
o It is responsible for spontaneous sterilization in
natural conditions - due to combination of
ultraviolet rays and heat.

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 PHYSICAL METHODS OF STERILIZATION
2. Heat
 Heat is considered to be most reliable method of
sterilization of articles that can withstand heat.
 Heat acts by oxidative effects as well as
denaturation and coagulation of proteins. Those
articles that cannot withstand high temperatures
can still be sterilized at lower temperature by
prolonging the duration of exposure.

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 PHYSICAL METHODS OF STERILIZATION
Factors affecting sterilization by heat are:
 Nature of heat: Moist heat is more effective than dry heat
 Temperature and time: are inversely proportional. As temperature
increases the time taken decreases.
 Number of microorganisms: More the number of
microorganisms, higher the temperature or longer the duration
required.
 Nature of microorganism: Depends on species and strain
 Spores are highly resistant to heat.

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 a) DRY HEAT:
i. Red heat: bacteriological loops, tips of forceps etc sterilized
by holding them in Bunsen flame till they become red hot.
Limited to articles that can be heated to redness in flame.
ii. Flaming: This is a method of passing the article over a
Bunsen flame, but not heating it to redness eg scalpels,
mouth of test tubes, glass slides and cover slips are passed
through the flame a few times.
iii. Incineration: destroying contaminated material by burning
in incinerator eg soiled dressings, pathological material and
bedding etc . This technique results in the loss of the article,
hence suitable only for those articles that have to be
disposed.

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iv. Hot air oven:
This method was introduced by Louis Pasteur. Articles to
be sterilized are exposed to high temp’ (160o C) for one
hour in an electrically heated oven. Even distribution of
heat throughout the chamber achieved by a fan.
Articles sterilized: Metallic instruments (forceps, scalpels,
scissors), glasswares (petri-dishes, pipettes, flasks, all-glass
syringes), swabs, and some pharmaceutical products.
Increasing temperature by 10 degrees shortens the
sterilizing time by 50 percent.
This is the only method of sterilizing oils and powders.

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v. Infra red rays
 Infrared rays bring about sterilization by generation
of heat.
 Articles to be sterilized are placed in a moving
conveyor belt and passed through a tunnel that is
heated by infrared radiators to a temperature of
180oC for 7.5 minutes.
 Articles sterilized include metallic instruments and
glassware.

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 b) MOIST HEAT
 Moist heat acts by coagulation and denaturation of
proteins.
At temperature below 100oC:
i. Pasteurization:
 This process was originally used by Louis Pasteur.
 Currently this procedure is used in food and dairy
industry. There are two methods of pasteurization,
the holder method (heated at 63oC for 30 minutes)
and flash method (heated at 72oC for 15 seconds)
followed by quickly cooling to 13oC.
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 PHYSICAL METHODS OF STERILIZATION
 Other pasteurization methods include Ultra-High
Temperature (UHT), 140oC for 15 sec and 149oC for
0.5 sec. This method is suitable to destroy most
milk borne pathogens like Salmonella,
Mycobacteria, Streptococci, Staphylococci and
Brucella, however Coxiella may survive
pasteurization. Others;
ii.Vaccine bath
iii. Serum bath
iv.Inspissation
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 At temperature 100oC:
i) Boiling
 Boiling water (100oC) kills most vegetative bacteria
and viruses immediately.
 Certain bacterial toxins such as Staphylococcal
enterotoxin are heat resistant.
 Some bacterial spores are resistant to boiling and
survive; hence this is not a substitute for
sterilization.
 When absolute sterility is not required, certain
metal articles and glasswares can be disinfected by
placing
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them
jimmy ongori 2013 in boiling water for 10-20 minutes.
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 ii) Steam at 100oC
 Free steam at 100oC, An autoclave can be used. A steamer is a metal
cabinet with perforated trays to hold articles.
 The bottom of steamer is filled with water and heated, steam generated
sterilizes the articles when exposed for a period of 90 minutes. Media eg
DCA and selenite broth sterilized by steaming.
 Sugar and gelatin in medium may get decomposed on autoclaving, hence they
are exposed to free steaming for 20 minutes for 3 successive days. This
process is known as tyndallisation (after John Tyndall) or fractional
sterilization or intermittent sterilization. The vegetative bacteria are killed in
the first exposure and the spores that germinate by next day are killed in
subsequent days. The success of process depends on the germination of
spores.
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At temperature above 100oC:
Autoclave:
 Sterilization can be effectively achieved at a temperature
above 100oC using an autoclave. Water boils at 100oC at
atmospheric pressure, but if pressure is raised, the
temperature at which the water boils also increases.
 In an autoclave the water is boiled in a closed chamber. As
the pressure rises, the boiling point of water also raises.
 At a pressure of 15 lbs inside the autoclave, the
temperature is said to be 121oC. Exposure of articles to
this temperature for 15 minutes sterilizes them.

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 To destroy the infective agents associated with
spongiform encephalopathies (prions), higher
temperatures or longer times are used; 135oC or
121oC for at least one hour are recommended.
Advantages of steam: It has more penetrative power
than dry air, it moistens the spores (moisture is
essential for coagulation of proteins
Articles sterilized: Culture media, dressings, certain
equipment, linen etc.
Disadvantages: Drenching and wetting of articles may
occur, trapped air may reduce the efficacy, takes long time
to cool
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 AUTOCLAVE

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RADIATION
 Two types of radiation are used, ionizing and non-ionizing.
 Non-ionizing rays are low energy rays with poor penetrative power
while ionizing rays are high-energy rays with good penetrative
power. Since radiation does not generate heat, it is termed "cold
sterilization".
i) Non-ionizing rays:
 Rays of wavelength longer than the visible light are non-ionizing.
 UV rays are generated using a high-pressure mercury vapor lamp.
 UV rays induce formation of thymine-thymine dimers, which
inhibits DNA replication.

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 RADIATION
 UV readily induces mutations in cells irradiated with a
non-lethal dose. Microorganisms such as bacteria, viruses,
yeast, etc. that are exposed to the effective UV radiation
are inactivated within seconds.
 UV rays don’t kill spores
 UV rays are used to disinfect hospital wards, operation
theatres, virus laboratories, corridors, etc. Disadvantages
- harmful to skin and eyes.

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 Ionizing Rays
 Used to sterilize articles like syringes, gloves,
dressing packs, foods and pharmaceuticals, petri
dishes, antibiotics, vitamins, hormones, glasswares
and fabrics.
 Sterilization is accomplished in few seconds.

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 FILTRATION
 Filtration does not kill microbes, it separates them
out.
 Membrane filters with pore sizes between 0.2-0.45
μm are commonly used to remove particles from
solutions that can't be autoclaved.
 Used to remove microbes from heat labile liquids
such as serum, antibiotic solutions, sugar solutions,
urea solution.
 Filtration is aided by using either positive or
negative pressure using vacuum pumps.
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Types of filters
1. Earthenware filters: These filters are made up of
diatomaceous earth or porcelain.
2. Asbestos filters
3. Sintered glass filters: These are made from finely
ground glass that are fused sufficiently to make small
particles adhere to each other.
4. Membrane filters
5. Air Filters: They are usually used in biological safety
cabinets.

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 CHEMICAL METHODS
 Disinfectants are those chemicals that destroy
pathogenic bacteria from inanimate surfaces. Some
chemical have very narrow spectrum of activity
and some have very wide. Those chemicals that
can sterilize are called chemisterilants. Those
chemicals that can be safely applied over skin and
mucus membranes are called antiseptics.

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 Agent Mechanisms of Action Comments
Surfactants Membrane Disruption; Soaps; detergents
Chemical Antimicrobials increased penetration
Quats (cationic Denature proteins; Antiseptic - benzalconium
detergent) Disrupts lipids chloride, Cepacol; Disinfectant
Organic acids High/low pH Mold and Fungi inhibitors; e.g.,
and bases benzoate of soda
Heavy Metals Denature protein Antiseptic & Disinfectant;
Silver Nitrate
Halogens Oxidizing agent Antiseptic - Iodine (Betadine)
Disrupts cell membrane Disinfectant - Chlorine (Chlorox)
Alcohols Denatures proteins; Antiseptic & Disinfectant
Disrupts lipids Ethanol and isopropyl
Phenolics Disrupts cell membrane Disinfectant
Irritating odor
Aldehydes Denature proteins Gluteraldehyde - disinfectant
(Cidex); Formaldehyde -
disinfectant
Ethylene Oxide Denaturing proteins Used in a closed chamber to
sterilize
Oxidizing agents Denature proteins Hydrogen peroxide – antiseptic;
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Benzoyl peroxide – antiseptic
Chemical Methods of Microbial Control
Types of Disinfectants
1. Phenols and Phenolics:
u Phenol (carbolic acid) was first used by Lister as a
disinfectant.
u Rarely used today because it is a skin irritant and
has strong odor.
u Phenolics are chemical derivatives of phenol,
Destroy plasma membranes and denature proteins.
u Cresols: eg Lysol
u Biphenols

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2. Halogens: Effective alone or in compounds.
A. Iodine:
u Tincture of iodine (alcohol solution) was one of first antiseptics
used, denatures proteins.
u Stains skin and clothes, somewhat irritating.
u Iodophors: Compounds with iodine that take several minutes to
act. Used as skin antiseptic in surgery. Not effective against
bacterial endospores eg; Betadine
B. Chlorine:
u When mixed in water forms hypochlorous acid, Used to
disinfect drinking water, pools, and sewage.
u Sodium hypochlorite - active ingredient of bleach.
u Chloramines: Consist of chlorine and ammonia. Less effective
as germicides.
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3. Alcohols:
u Kill bacteria, fungi, but not endospores or naked
viruses.
u Act by denaturing proteins and disrupting cell
membranes. Evaporate, leaving no residue.
u Used to mechanically wipe microbes off skin before
injections or blood drawing.
u Not good for open wounds, because cause proteins
to coagulate.
u Ethanol: Optimum concentration is 70%.
u Isopropanol: Better disinfectant than ethanol.
Also cheaper and less volatile.
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4. Heavy Metals:
u Include copper, selenium, mercury, silver, and zinc.
A. Silver: 1% silver nitrate used to protect infants against
gonorrheal eye infections until recently.
B. Copper; Copper sulfate is used to kill algae in pools and
fish tanks.
C. Selenium: Kills fungi and their spores. Used for fungal
infections. Also used in dandruff shampoos.
E. Zinc: Zinc chloride is used in mouthwashes.
u Zinc oxide is used as antifungal agent in paints.

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5. Quaternary Ammonium Compounds:
u Widely used surface active agents.
u Effective against gram +VE bacteria, less effective
against gram -VE bacteria. Also destroy fungi,
amoebas, and enveloped viruses.
u Zephiran, Cepacol
u Advantages: Strong antimicrobial action, colorless,
odorless, tasteless, stable, and nontoxic.
u Diasadvantages: Form foam. Organic matter
interferes with effectiveness. Neutralized by soaps
and anionic detergents.

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6. Aldehydes:
Include some of the most effective antimicrobials.
Inactivate proteins by forming covalent crosslinks
with several functional groups.
A. Formaldehyde gas:
Excellent disinfectant.
Commonly used as formalin
Formalin is used extensively to preserve biological
specimens and inactivate viruses and bacteria in
vaccines.
Irritates mucous membranes, strong odor.
Also used in mortuaries for embalming.
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B. Glutaraldehyde:
Less irritating and more effective than formaldehyde.
One of the few chemical disinfectants that is a
sterilizing agent.
A 2% solution of glutaraldehyde (Cidex) is:
Bactericidal, tuberculocidal, and viricidal in 10
minutes.
Sporicidal in 3 to 10 hours.
Commonly used to disinfect hospital instruments.
Also used in mortuaries for embalming.

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7. Gaseous Sterilizers:
Chemicals that sterilize in a chamber similar to an
autoclave.
A. Ethylene Oxide:
Kills all microbes and endospores, but requires
exposure of 4 to 18 hours.
Toxic and explosive in pure form.
Highly penetrating.
Many hospitals have ethylene oxide chambers to
sterilize mattresses and large equipment.

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8. Peroxygens (Oxidizing Agents):
Oxidize cellular components of treated microbes.
Disrupt membranes and proteins.
A. Ozone:
Highly reactive form of oxygen.
Used along with chlorine to disinfect water.
Helps neutralize unpleasant tastes and odors.
More effective killing agent than chlorine, but less
stable and more expensive.
Made by exposing oxygen to electricity or UV light.

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B. Hydrogen Peroxide:
Used as an antiseptic.
Not good for open wounds because quickly broken
down by catalase present in human cells.
Effective in disinfection of inanimate objects.
Sporicidal at higher temperatures.
C. Benzoyl Peroxide:
Used in acne medications.

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 D. Peracetic Acid:
One of the most effective liquid sporicides available.
Kills bacteria and fungi in less than 5 minutes.
Kills endospores and viruses within 30 minutes.
Used widely in disinfection of food and medical
instruments because it does not leave toxic residues.

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 BACTERIA

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• The bacteria (singular: bacterium) are a large group of
unicellular microorganisms. Typically a few micrometres in length,
bacteria have a wide range of shapes, ranging from spheres to rods
and spirals.
• Bacteria are ubiquitous in every habitat on Earth, growing in soil,
acidic hot springs, radioactive waste, water, and deep in the Earth's
crust, as well as in organic matter and the live bodies of plants and
animals.
• There are typically 40 million bacterial cells in a gram of soil and a
million bacterial cells in a millilitre of fresh water
• Bacteria are vital in recycling nutrients, with many steps in nutrient
cycles depending on these organisms, such as the fixation of
nitrogen
• The study of bacteria is known as bacteriology, a branch of
122 jimmy ongori 2013 03/11/2013 21:18
microbiology.
• There are about ten times as many bacterial cells in the
human flora of bacteria as there are human cells in the
body, with large numbers of bacteria on the skin and as gut
flora.
• Majority of the bacteria in the body are rendered harmless
by the protective effects of the immune system, and a few
are beneficial. A few species of bacteria are pathogenic and
cause infectious diseases.
• Bacteria are important in sewage treatment, the
production of cheese and yoghurt through fermentation, as
well as in biotechnology, and the manufacture of antibiotics
and other chemicals.
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• Once regarded as plants, bacteria are now classified as
prokaryotes. Unlike cells of animals and other eukaryotes,
bacterial cells do not contain a nucleus and rarely harbour
membrane-bound organelles.
• Although the term bacteria traditionally included all
prokaryotes, the scientific classification changed after the
discovery in the 1990s that prokaryotes consist of two very
different groups of organisms that evolved independently
from an ancient common ancestor.
• These are called Bacteria and Archaea.
• Bacteria were first observed by Antonie van Leeuwenhoek
in 1676, using a single-lens microscope of his own design.
He called them "animalcules"
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• The name bacterium was introduced much later, by Christian
Gottfried Ehrenberg in 1838.
• Louis Pasteur demonstrated in 1859 that the fermentation
process is caused by the growth of microorganisms, and that
this growth is not due to spontaneous generation. (Yeasts and
molds, commonly associated with fermentation, are fungi.)
• Along with Robert Koch, Pasteur was an early advocate of the
germ theory of disease. Robert Koch was a pioneer in medical
microbiology and worked on cholera, anthrax and tuberculosis.
In his research into tuberculosis, Koch finally proved the germ
theory. In Koch's postulates, he set out criteria to test if an
organism is the cause of a disease these postulates are still used
today.
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PROKARYOTES AND EUKARYOTES
• "True" bacteria (which include all bacteria that
infect man) are members of one kingdom (the
eubacteria).
• A group of organisms often found in extreme
environments form a second kingdom
(archaebacteria, Archaea).
• Morphologically, the two kingdoms of organisms
appear similar, especially in the absence of a nucleus,
and thus are classified together as prokaryotes.
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PROKARYOTES AND EUKARYOTES
• However, they have major biochemical differences.
Most archaea live in environments such as hot sulfur
springs where they experience temperatures as high
as 800 C and a pH 2. - called thermoacidophiles.
Others live in methane-containing (methanogens) or
high salt (extreme halophiles) environments.
• Members of the Archaea are not human
pathogens

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 SUMMARY OF DIFFERENCES
PROKARYOTIC CELLS EUKARYOTIC CELLS

• Small cells (<5 μm) • Larger cells (>10 μm)

• Always unicellular • Often multicellular

• No nucleus or any membrane-bound • Always have nucleus and other

organelles membrane-bound organelles

• DNA is circular, without proteins • DNA is linear and associated with

proteins to form chromatin

• Ribosomes are small (70S) • Ribosomes are large (80S)

• No cytoskeleton • Always has a cytoskeleton

• Cell division is by binary fission • Cell division is by mitosis or meiosis

• 128Reproduction
jimmy ongoriis always asexual
2013 • Reproduction is asexual or sexual
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 EUKARYOTIC CELL

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 THE PROTOTYPE BACTERIAL CELL

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 ASSIGNMENT
 Draw a diagram of a Gram
negative and Gram positive
bacterial cell wall and briefly
describe the differences

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BACTERIAL STRUCTURES
• Not all bacteria possess all of these components.
1. The cell envelope
 Bacteria can be divided into two groups on the basis of
staining with the Gram stain; Gram positive bacteria
remain stained by crystal violet on washing, Gram negative
do not.
 All bacteria have a cell membrane where oxidative
phosphorylation occurs (since there are no mitochondria).
Outside the cell membrane is the cell wall which is rigid
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and protects the cell from osmotic lysis.
 In Gram +ve bacteria, the cell wall peptidoglycan layer is much
thicker than in Gram -ve bacteria. Gram -ve bacteria have an
additional outer membrane. The outer membrane is the major
permeability barrier in Gram negative bacteria.
 The space between the inner and outer membranes is known as the
periplasmic space. Gram -ve bacteria store degradative enzymes in
the periplasmic space.
 Gram +ve bacteria lack a periplasmic space; instead they secrete
exoenzymes and perform extracellular digestion.
 Digestion is needed since large molecules can not readily pass
across the outer membrane (if present) or cell membrane.

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THE BACTERIAL CELL WALL
• As in other organisms, the bacterial cell wall
provides structural integrity to the cell. In
prokaryotes, the primary function of the cell wall is
to protect the cell from internal turgor pressure
caused by the much higher concentrations of
proteins and other molecules inside the cell
compared to its external environment. The bacterial
cell wall differs from that of all other organisms by
the presence of peptidoglycan, which is located
immediately outside of the cytoplasmic membrane.
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THE BACTERIAL CELL WALL
• Peptidoglycan is responsible for the rigidity of the
bacterial cell wall and for the determination of cell
shape
• All bacterial cell walls (with a few exceptions e.g.
Mycoplasma) contain peptidoglycan, not all cell walls
have the same overall structures.
• There are two main types of bacterial cell walls,
Gram positive and Gram negative, which are
differentiated by their Gram staining characteristics.
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TYPES OF BACTERIAL CELL ENVELOPES
Mycobacteria
• The Mycobacteria have a cell envelope which is not
typical of Gram positives or Gram negatives.
• The mycobacterial cell envelope does not consist of
the outer membrane characteristic of Gram
negatives, but has a significant peptidoglycan-
arabinogalactan-mycolic acid wall structure which
provides an external permeability barrier.

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The Gram positive cell wall
• The Gram positive cell wall has a very thick peptidoglycan layer, which is
responsible for the retention of the crystal violet dyes during the Gram
staining procedure.
The Gram negative cell wall
• It contains a thin peptidoglycan layer adjacent to the cytoplasmic
membrane. This is responsible for the cell wall's inability to retain the
crystal violet stain upon decolourisation with ethanol during Gram
staining.
• In addition to the peptidoglycan layer, the Gram negative cell wall also
contains an outer membrane composed by phospholipids and
lipopolysaccharides whose chemical structure is often unique to specific
bacterial strains (i.e. sub-species) and is responsible for many of the
antigenic properties of these strains.
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Plasmids
 These are extra-chromosomal DNA, usually present in multiple copies,
that often code for pathogenesis factors and antibiotic resistance factors.
Some forms are also involved in bacterial replication.
Flagella
 Some bacterial species are mobile and possess locomotory organelles –
flagella. Those that do are able to taste their environment and respond to
specific chemical foodstuffs or toxic materials and move towards or away
from them (chemotaxis).
 Flagella are embedded in the cell membrane, extend through the cell
envelope and project as a long strand. They move the cell by rotating
with a propeller like action.

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Pili (synonym: fimbriae)
 The types of pili varies both among and between species. Pili are hair-
like projections of the cell.
 Some are involved in sexual conjugation and others allow adhesion
to host epithelial surfaces in infection.
Capsules and slime layers
 Surround the outside of the cell envelope. When more defined, they are
referred to as a capsule when less defined as a slime layer or glycocalyx.
 usually consist of polysaccharide; however, in certain bacilli they are
composed of a polypeptide .
 Capsules of pathogenic bacteria inhibit ingestion and killing by
phagocytes.

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Endospores (spores)
(Read and make notes on spores)
 These are a dormant form of a bacterial cell produced by
certain bacteria when starved; the actively growing form
of the cell is referred to as vegetative. The spore is resistant
to adverse conditions (including high temperatures and
organic solvents).
 The spore cytoplasm is dehydrated and contains calcium
dipicolinate which is involved in the heat resistance of the
spore.
 Spores are commonly found in the genera Bacillus and
Clostridium.
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CELL MORPHOLOGY
• Bacteria display a wide diversity of shapes and sizes, called
morphologies.
• Bacterial cells are about one tenth the size of eukaryotic cells and are
typically 0.5–5.0 μm in length.
• A few species eg Thiomargarita namibiensis – are up to half a millimetre
long and are visible to the unaided eye.
• Among the smallest bacteria are members of the genus Mycoplasma,
which measure only 0.3 micrometres.
• Most bacterial species are; spherical, called cocci ( Greek
kókkos, grain/seed) or rod-shaped, called bacilli (Latin baculus, stick).
• Some rod-shaped bacteria, called vibrio, are slightly curved or comma-
shaped; others, can be spiral-shaped, called spirilla, or tightly coiled,
called spirochaetes.
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CELL MORPHOLOGY Cont’
• This wide variety of shapes is determined by the bacterial cell wall
and cytoskeleton
• Many bacterial species exist as single cells, others associate in
characteristic patterns:
• Neisseria form diploids (pairs)
• Streptococcus form chains
• Staphylococcus group together in "bunch of grapes" clusters.
• Bacteria can also be elongated to form filaments, for example the
Actinobacteria. Filamentous bacteria are often surrounded by a
sheath that contains many individual cells.
• Bacteria are classified by direct examination with the light
microscope through its morphology and aggregation.
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COCCI (singular - coccus)
 Are any bacteria whose overall shape is spherical or nearly
spherical. Describing a bacterium as a coccus, or sphere,
distinguishes it from bacillus, or rod. This is the first of many
taxonomic traits for identifying and classifying a bacterium
• Basic forms;
1. Pairs – diplococci eg Neisseria gonorrhoeae
2. Groups of 4 or 8 known as tetrads or sarcina eg Micrococci
3. Bead-like chains, or streptococci eg Streptococcus
pneumoniae
4. Grapelike clusters, or staphylococci eg Staphylococcus
aureus
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Bacillus
 Although Bacillus refers to the genus, the word bacillus may
also be used to describe any rod-shaped bacterium, and in this
sense, bacilli are found in many different taxonomic groups of
bacteria.
 Bacilli are usually solitary, but can combine to form
diplobacilli, streptobacilli, and palisades.
Coccobacillus
• A type of rod-shaped bacteria. The word coccobacillus reflects an
intermediate shape between coccus and bacillus.
• Coccobacilli rods are so short and wide that they resemble cocci.
Haemophilus influenzae and Chlamydia trachomatis are
coccobacilli.
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145 jimmy ongori 2013 03/11/2013 21:18
 PATHOGENESIS
OF
BACTERIAL INFECTION

PATHOGENICITY TOXIGENICITY
VIRULENCE

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 The pathogenesis of bacterial infection includes the
initiation of the infectious process and the mechanisms
leading to the development of signs and symptoms of
bacterial disease.
 The outcome of the interaction between bacteria and
host is determined by characteristics that favour
establishment of the bacteria within the host and their
ability to damage the host as they are opposed by host
defense mechanisms.
 Among the characterics of bacteria are;
 Adherence to host cells
 Invasiveness
 Toxigenity
 Ability to evade the host´s immune system.
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Pathogenesis of bacterial infection
 Humans and animals have abundant normal microflora.
 Most bacteria do not produce disease but achieve a balance
with the host that ensures the survival, growth, and
propagation of both the bacteria and the host.
 Sometimes bacteria are pathogenic (e.g. Salmonella typhi)
but infection remains latent or subclinical and the host
is a "carrier" of the bacteria.
 Analysis of infection and disease through the application of
principles such as Koch´s postulates leads to
classification
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of bacteria as pathogenic or non-pathogenic.
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 Some bacterial species are always considered to be
pathogens, and their presence is abnormal. Eg
Mycobacterium tuberculosis and Yersinia pestis (plague).
 Some sp. are part of the normal flora of humans but
can cause disease. Eg Escherichia coli is part of the
GIT flora of normal humans, but it is also a comon
cause of urinary tract infection and traveller´s
diarrhea,

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The infectious process
 Infection indicates multiplication of M.O.s
 Prior to multiplication, bacteria must enter and
establish themselves within the host.
 The most frequent portals of entry;
 the respiratory (mouth and nose)
 gastrointestinal
 urogenital tracts
 abnormal areas of mucous membranes and skin (e.g.
cuts, burns) are also frequent sites of entry.

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 The infectious process
 Once in the body, bacteria must attach or adhere
to host cells - usually epithelial cells.
 After the bacteria have established a primary site
of infection, they multiply and spread.
 Infection can spread directly through tissues or via
the lymphatic system to bloodstream.
 Bloodstream infection is known as bacteremia.
 Bacteremia allows bacteria to spread widely in the
body and permits them to reach tissues suitable for
their multiplication.
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 TERMINOLOGIES
 Infection:
 Multiplication of an infectious agent within the body.
 Multiplication of the bacteria that are part of normal
flora is generally not considered an infection.
 Pathogenicity:
 The ability of an infectious agent to cause disease.
 Virulence:
 The degree or extent of pathogenicity
 The quantitative ability of an agent to cause disease.
 Virulence involves invasiveness and toxigenicity.
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 Virulence factors
 These are traits or features that allow or enhance
the microorganism’s ability to cause disease,
include;
i. adhesion organelles,
ii. toxin production
iii. evasion of the host’s immune response
iv. resistance to antibiotics
v. ability to invade host tissues
vi. enhanced intracellular survival and growth
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 Toxigenicity:
 ability of a microorganism to produce a toxin that
contributes to the development of disease.
 Invasion:
 The process whereby bacteria, parasites, fungi and
viruses enter the host cells or tissues and spread in
the body.
 Pathogen:
 A microorganism capable of causing disease.
 Opportunistic pathogen:
 An agent capable of causing disease only when the
host´s resistance is impaired (e.g. the patient is
immunocompromised).
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 Bacterial virulence factors

1. TOXINS
 Toxins produced by bacteria are generally classified
into two groups:

 Exotoxins

 Endotoxins - Only found in gram negative bacteria

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Endotoxins of gram-negative bacteria
 Endotoxins are toxic components of the bacterial cell
envelope.
 The classical and most potent endotoxin is
lipopolysaccharide derived from bacterial cell walls and are
liberated when the bacteria lyse.
 The pathophysiologic effects of LPS are similar
regardless of their bacterial origin
 Relatively stable; withstand heating at temperatures above
60°C for hours without loss of toxicity
 Usually produce fever in the host
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 Exotoxins
 Many gram-positive and gram-negative bacteria produce
exotoxins of considerable medical importance - that
modify, by enzymatic action, or destroy certain cellular
structures.
 Examples - botulism, anthrax, cholera and diphtheria.
 Vaccines have been developed for some of the exotoxin-
mediated diseases and continue to be important in the
prevention of disease.
 These vaccines—called toxoids—are made from
exotoxins, which are modified so that they are no longer
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 1. Diphtheria toxin (Corynebacterium diphtheriae)
 It is a Gm +ve rod that can grow on the mucous
membranes of the upper respiratory tract or in minor
skin wounds. Some strains produce diphtheria toxin.
2. Tetanospasmin (toxin of Clostridium tetani)
 C. tetani is an anaerobic gram-positive rod
 It contaminates wounds, and the spores germinate in the
anaerobic environment of the devitalized tissue. The
vegetative forms of Clostridium tetani produce toxin
tetanospasmin.
 Toxin reaches the CNS and acts by blocking release of an
inhibitory mediator in motor neuron synapses.
 Extremely small amount of toxin can be lethal for
158 humans.
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3. Botulotoxin (toxin of Clostridium botulinum)
 Clostridium botulinum is found in soil or water and
may grow in foods if the environment is
appropriately anaerobic.
 It is the most potent toxin known – causes botulism
 It is heat-labile and is destroyed by sufficient heating.
 Toxin is absorbed from the GIT and carried to
motor nerves, where it blocks the release of
acetylcholine at synapses and neuromuscular
junctions. Muscle contraction does not occur, and
paralysis results.
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4. Toxins of Clostridium perfringens
 Spores of Clostridium perfringens are introduced into
the wounds by contamination with soil or faeces. In
the presence of necrotic tissue, spores germinate
and vegetative cells produce several different
toxins.
 Many are necrotizing and hemolytic and favour the
spread of gangrene

5. Streptococcal erythrogenic toxin

 Some strains of hemolytic streptococci produce a
toxin that results in a rash, as in scarlet fewer.
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6. Toxic shock syndrom toxin - 1 (TSST-1)
 Some Staphylococcus aureus strains growing on mucous
membranes (e.g. on the vagina in association with
menstruation), or in wounds, produce TSST-1.
 Toxic shock syndrome is characterized by shock, high
fewer, and a diffuse red rash
7. Exotoxins associated with diarrheal diseases
 Vibrio cholerae toxin
 Staphylococcus aureus enterotoxin
 Other enterotoxins are produced; Yersinia enterocolitica,
Vibrio parahaemolyticus, Aeromonas species
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 Enzymes
 Many bacteria produce enzymes that play an
important role in the infectious process;
i. Collagenase: degrades collagen, the major protein of
fibrous connective tissue, and promotes spread of
infection in tissue.
ii. Coagulase: Staphylococccus aureus produce coagulase,
which coagulates plasma. Coagulase contributes to the
formation of fibrin walls around staphylococcal lesions,
which helps them persist in tissues.
iii. Hyaluronidases: hydrolyze hyaluronic acid, a
constituent of substance of connective tissue (e.g.
staphylococci, streptococci and anaerobes) and aid in
162 their spread
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 Enzymes
iv. Streptokinase: many hemolytic streptococci produce
streptokinase (fibrinolysin), a substance that activates a
proteolytic enzyme of plasma. It dissolves coagulated
plasma and aids in the spread of streptococci through
tissues. Streptokinase is used in treatment of acute
myocardial infarction to dissolve fibrin clots.
v. Hemolysins and leukocidins: Many bacteria produce
substances that are cytolysins - they dissolve red blood
cells (hemolysins) or kill tissue cells or leukocytes
(leukocidins).

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Antiphagocytic factors
 Many bacterial pathogens are rapidly killed once they are
ingested by polymorphonuclear cells or macrophages.
 Some pathogens evade phagocytosis or leukocyte
microbidical mechanisms e.g. Streptococcus pneumoniae
have polysaccharide capsules.
Adherence factors
 Once bacteria enter the body of the host, they must
adhere to cells of a tissue surface. If they do not adhere,
they would be swept away by mucus and other fluids
 Adherence is followed by development of microcolonies
and subsequent complex steps in the pathogenesis of
infection.
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 STAINING

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 Staining
Staining is a technique used in microscopy to
enhance contrast in the microscopic image.
Stains and dyes are frequently used in biology and
medicine to highlight structures in biological tissues
for viewing, often with the aid of different
microscopes.
Stains may be used to define and examine bulk
tissues (highlighting, muscle fibers or connective
tissue), classifying different blood cells.

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 Staining
In biochemistry it involves adding a class-specific
(DNA, proteins, lipids, carbohydrates) dye to a
substrate to qualify or quantify the presence of a
specific compound
Biological staining is also used to mark cells in flow
cytometry, and in gel electrophoresis.
In vivo staining is the process of dyeing living tissues
By causing certain cells or structures to take on
contrasting colours, their morphology or position
within a cell can be distinguished
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 Staining
In vitro staining involves colouring cells or structures that
are no longer living. Certain stains are often combined to
reveal more details and features than a single stain alone.
A counter-stain is a stain that makes cells or structures
more visible, when not completely visible with the
principal stain.
For example, crystal violet stains only Gram +ve bacteria
during Gram staining. A safranin counterstain is applied
which stains all cells, allowing the identification of Gram
-ve bacteria .

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 GRAM STAINING
The method is named after its inventor, Hans Christan Gram
(1853–1938)
Gram staining is a bacteriological laboratory technique
used to differentiate bacterial species into two large groups
(Gram +ve and Gram -ve) based on the physical properties
of their cell walls
In a modern molecular microbiology lab, most
identification is done using genetic sequences and other
molecular techniques, which are more specific than
differential staining.
169
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 The Gram stain is the most widely used staining
procedure in bacteriology.
 It is called a differential stain since it
differentiates between Gram+VE and Gram-VE
negative bacteria.
 Bacteria that stain purple with the Gram staining
procedure are termed Gram +VE; those that
stain pink are said to be Gram -VE. The terms
have nothing to do with electrical charge, but
simply designate two distinct morphological
groups of bacteria.
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 Gram-positive and Gram-negative bacteria
stain differently because of fundamental
differences in the structure of their cell
walls.
 The bacterial cell wall serves to give the
organism its size and shape as well as to
prevent osmotic lysis. The material in the
bacterial cell wall which confers rigidity is
peptidoglycan.

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 Principles Gram Staining 172

Gram stains are performed on body fluid or biopsy

 Gram staining tests the bacterial cell wall's ability to retain
crystal violet dye during solvent treatment.
 Safranin is added as a mordant to form the crystal
violet/safranin complex in order to render the dye
impossible to remove.
 Ethyl-alcohol solvent acts as a decolorizer and dissolves
the lipid layer from gram-negative cells. This enhances
leaching of the primary stain from the cells into the
surrounding solvent.
 Ethyl-alcohol will dehydrate the thicker gram-positive cell
walls, closing the pores as the cell wall shrinks.
 For this reason, the diffusion of the crystal violet-safranin
staining is inhibited,
jimmy ongori 2013 so the bacteria remain stained.
03/11/2013 21:18
 STAINING MECHANISM
Gram +ve bacteria have a thick mesh-like cell wall made
of peptidoglycan (50-90% of cell wall), which are stained
purple by crystal violet
Gram -ve bacteria have a thinner layer (10% of cell wall),
which are stained pink by the counter-stain. There are four
basic steps of the gram stain:
1. Applying a primary stain (crystal violet) to a heat-
fixed (death by heat) smear
2. The addition of a trapping agent (gram's iodine)
3. Rapid decolorization with alcohol or acetone
4. Counterstaining with safranin.
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 PROCEDURE FOR G/STAIN
(1) Fix smear by heat.
(2) Cover with crystal violet for 30-60 seconds
(3) Wash with water. Do not blot.
(4) Cover with Gram's iodine for 30-60 seconds
(5) Wash with water. Do not blot.
(6) Decolorize for 10–30 seconds with gentle
agitation in acetone-alcohol

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(7) Wash with water. Do not blot.
(8) Cover for 10–30 seconds with safranin (2.5%
solution in 95% alcohol).
(9) Wash with water and let dry.
(10) Examine under oil immersion objective
 Gram-positive bacteria stain dark blue or violet
 Gram-negative organisms will appear red or pink
because they are counterstaine

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 Step 1. Staining with crystal violet.
 Step 2. Fixation with iodine stabilizes crystal violet
staining. All bacteria remain purple or blue.
 Step 3. Extraction with alcohol or other solvent.
Decolorizes some bacteria (Gram negative) and not
others (Gram positive).
 Step 4. Counterstaining with safranin. Gram positive
bacteria are already stained with crystal violet and
remain purple. Gram negative bacteria are stained
pink.

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177 jimmy ongori 2013 03/11/2013 21:18
 ZIEHL–NEELSEN STAIN
The Ziehl–Neelsen stain, also known as the acid-fast
stain, was first described by two German doctors;
Franz Ziehl (1859 to 1926), and Friedrich Neelsen
(1854 to 1898)
It is a special bacteriological stain used to identify
acid-fast organisms, mainly Mycobacteria.
It is helpful in diagnosing M. tuberculosis since its
lipid rich cell wall makes it resistant to Gram stain.
 The reagents used are Ziehl–Neelsen carbolfuchsin,
acid alcohol and methylene blue. Acid-fast bacilli will
be bright red after staining.
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178
 Acid-Fast Principles
 Primary stain penetrates cell wall
 Intense decolourization does not release
primary stain from the cell wall of AFB
 Colour of AFB-based on primary stain
 Counterstain provides contrasting background

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 PROCEDURE
1. Heat fix the smear
2. Cover with carbol fuchsin stain
3. Heat the until vapour just begins to rise, do not
overheat. Allow the heated stain to remain on the
slide for 5 minutes
4. Wash off the stain with clean water
5. Cover the stain with 3% acid alcohol for 5 minutes
or until the stain is fully decolorized ie pale pink

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jimmy ongori 2013
 Procedure cont’
6. Wash with clean water
7. Cover the smear with methylene blue counter stain
for 1 minute
8. Wash with clean water
9. Air dry on a rack
10. Examine using 100X oil immersion objective

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 THE COCCI

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 COCCI
 The cocci are one of the most commonly encountered
bacteria (the other being bacilli)
 Cocci refers to the spherical shape
 Can exist as;
 Single cells
 Diplococcus eg Neisseria
 Long chains – Streptococcus, Enterococcus and
Lactococcus
 Irregular clumps – Staphyloccus
 Tetrads – genus Micrococcus
 Cubical packets of 8 cells – genus Sarcina

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 STAPHYLOCCUS
 Are gram-positive spherical cells, usually arranged in
grape-like irregular clusters.
 Some are members of the normal flora of the skin and
mucous membranes of humans; others cause suppuration,
abscess formation, a variety of pyogenic infections, and
even fatal septicaemia.
 Pathogenic staphylococci often haemolyse blood, coagulate
plasma, and produce a variety of extracellular enzymes and
toxins.
 The most common type of food poisoning is caused by a
heat-stable staphylococcal enterotoxin.
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 Staphylococci rapidly develop resistance to many
antimicrobial agents
 The genus Staphylococcus has at least 35 species.
 The three main species of clinical importance are
S. aureus, S. epidermidis, and S. saprophyticus.
 S aureus is a major pathogen for humans. Almost
every person will have some type of S aureus
infection during a lifetime, ranging in severity from
food poisoning or minor skin infections to severe
life-threatening infections.

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General Characteristics
 Gram-positive cocci, nonmotile,
 Facultative anaerobes
 Cells occur in grapelike clusters
 Do not form spores but may have capsules
 Salt-tolerant: allows them to tolerate the salt
present on human skin
 Tolerate desiccation: allows survival on
environmental surfaces (fomites)

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 STAPHYLOCOCCUS AUREUS
 Many neonates, most children and adults become
transiently colonized by S. aureus.
 The organism is carried in the nasopharynx,
occasionally on their skin and clothing and more
rarely in the vagina, in the rectum and or perineal
area.
 One of the commoner causes of opportunistic
infections in the hospital and community; including
pneumonia, osteomyelitis, septic arthritis,
bacteremia, endocarditis, abscesses/boils and other
skin infections.
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 Virulence factors of S. aureus
Enzymes:
 Coagulase – coagulates plasma and blood; produced
by 97% of human isolates; it is diagnostic
 Hyaluronidase – digests connective tissue
 Staphylokinase – digests blood clots
 DNase – digests DNA
 Lipases – digest oils; enhances colonization on skin
 Penicillinase – inactivates penicillin (very important
in resistance to penicillins)
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 Virulence factors of S. aureus
Toxins:
 Hemolysins – lyse red blood cells
 Leukocidin – lyses neutrophils and macrophages
 Enterotoxin – induce gastrointestinal distress
 Exfoliative toxin – separates the epidermis from
the dermis
 Toxic shock syndrome toxin (TSST) – induces fever,
vomiting, shock, systemic organ damage

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 Epidemiology
 Present in most environments frequented by
humans
 Readily isolated from fomites
 Carriage rate for healthy adults is 20-60%, mostly in
anterior nares, skin, nasopharynx, intestine
 Predisposition to infection include: poor hygiene
and nutrition, tissue injury, pre-existing primary
infection, diabetes, immunodeficiency
 Increase in community acquired methicillin
resistance - MRSA
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 Staphylococcal Disease
1. Localized cutaneous infections
 Folliculitis – superficial inflammation of hair follicle
 Furuncle – boil; inflammation of hair follicle or
sebaceous gland progresses into abscess/pustule
 Carbuncle – larger and deeper lesion created by
aggregation and interconnection of a cluster of
furuncles
 Impetigo – bubble-like swellings that can break and
peel away; most common in newborns
 Abscess - Predominant pathogen in about 50% of skin
abscesses
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 2. Systemic Disease
i. Toxic shock syndrome - TSS toxin is absorbed into
the blood and causes shock. Toxic shock syndrome
is manifested by onset of high fever, vomiting,
diarrhea, myalgias, rash, and hypotension with
cardiac and renal failure in the most severe cases.
 Occurs within 5 days after the onset of menses in
young women who use tampons, but can occur in
children or in men with staphylococcal wound
infections. TSS -associated S aureus can be found
in the vagina, on tampons, in wounds or other
localized infections, or in the throat but virtually
192 never in2013the bloodstream.
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 • TSS -associated S aureus can be found in the
vagina, on tampons, in wounds or other
localized infections, or in the throat but
virtually never in the bloodstrea
ii. Bacteremia - presence of bacteria in the
blood
iii.Endocarditis - occurs when bacteria attack
the lining of the heart
iv.Pneumonia - inflammation of the lungs
v. Osteomyelitis - inflammation of the bone
marrow and the surrounding bone
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 STAPHYLOCOCCAL FOLLICULITIS

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 STYE

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 STAPHYLOCOCCAL FURUNCLE - “ BOIL”

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 Staphylococcal Scalded Skin
Syndrome.

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 Staph skin infections
Furuncle
Deep folliculitis (infected hair follicle

superficialfolliculitis

Carbuncle
Multiple abcesses
Around many hair
follicles

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Scalded skin
Staph impetigo syndrome
Diagnostic Laboratory Tests
 Specimens - pus, blood, tracheal aspirate, or spinal
fluid for culture, depending upon the localization of
the process.
 Smears - appear as gram positive cocci in clusters in
Gram-stained smears of pus or sputum.
 Culture - Specimens planted on blood agar plates
give rise to typical colonies in 18 hours at 37 °Cs.

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 Clinical Concerns and Treatment
 95% have penicillinase and are resistant to
penicillin and ampicillin
 MRSA – methicillin-resistant S. aureus – carry
multiple resistance
 Some strains have resistance to all major drug
groups except vancomycin
 Abscesses have to be surgically perforated
 Systemic infections require intensive lengthy
therapy
 Amoxil/cloxacilin, vancomicin
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 Prevention

 Hand antisepsis is the most important measure

in preventing nosocomial infections
 Also important is the proper cleansing of
wounds and surgical openings, aseptic use of
catheters or indwelling needles, an appropriate
use of antiseptics

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Staphylococcus epidermidis
 Staphylococcus epidermidis is a less common cause of
opportunistic infections than S. aureus, but is still
significant. It is a mediator of nosocomial infections (e.g.
catheters, shunts, surgery). It is a major component of the
skin flora
Staphylococcus saprophyticus
 This organism is a significant cause of urinary tract
infections. It is also coagulase-negative and is not usually
differentiated from S. epidermidis clinically.

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 STREPTOCOCCI

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 General Characteristics of Streptococci
 Gram-positive, spherical arranged in long chains;
commonly in pairs
 Non-spore-forming, nonmotile
 Can form capsules and slime layers
 Facultative anaerobes
 Most parasitic forms are fastidious and require
enriched media
 Small, non-pigmented colonies on culture
 Sensitive to drying, heat, and disinfectants

205 jimmy ongori 2013 03/11/2013 21:18

 Are subdivided into groups by antibodies that recognize
surface antigens.
 The most important groupable streptococci are A, B and D.
 Three types of hemolysis reaction (alpha, beta, gamma)
are seen after growth of streptococci on sheep blood agar.
 Alpha refers to partial hemolysis with a green coloration,
Beta refers to complete clearing and gamma means there
is no lysis.
 Group A and group B streptococci are beta hemolytic,
whilst D are usually alpha or gamma.
 Streptococcus pneumoniae and viridans ("green")
streptococci are alpha hemolytic.
 Thus, the hemolysis reaction is important in grouping
jimmy ongori 2013
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 Human Streptococcal Pathogens
 S. pyogenes
 S. agalactiae
 Viridans streptococci
 S. pneumoniae
 Enterococcus faecalis

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β-Hemolytic S. pyogenes -
 Pyogenes means pus producing
 Individual cocci are spherical or ovoid and are
arranged in chains.
 Are non-motile
 Most group A strains produce capsules - impede
phagocytosis.
 Are Lancefield Serological Group A
 Have hair-like pili projecting through the capsule -
important in the attachment of streptococci to
epithelial cells
 It is the most serious streptococcal pathogen
 Strict parasite
 Inhabits throat, nasopharynx, occasionally skin
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 Virulence Factors
Extracellular toxins:
 Streptolysins – hemolysins; streptolysin O (SLO) and
streptolysin S (SLS) – both cause cell and tissue injury
 Erythrogenic toxin (pyrogenic) – induces fever and
typical red rash
 Superantigens – strong monocyte and lymphocyte
stimulants; cause the release of tissue necrotic factor
Extracellular enzymes:
 Streptokinase (fibrinolysin) – digests fibrin clots
 Hyaluronidase – breaks down connective tissue
 DNase – hydrolyzes DNA
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 Epidemiology
 Humans are the only reservoir
 Inapparent carriers
 Transmission – contact, droplets, food, fomites
 Portal of entry - skin or pharynx
 Children predominant group affected for cutaneous
and throat infections
 Systemic infections and progressive sequelae
possible if untreated

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PATHOGENICITY

1. Skin infections
 Impetigo (pyoderma) – superficial lesions that
break and form highly contagious crust; often
occurs in epidemics in school children
 Erysipelas – pathogen enters through a break in
the skin and eventually spreads to the dermis and
subcutaneous tissues; can remain superficial or
become systemic

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2. Throat infections
Streptococcal pharyngitis
 The most common infection due to - hemolytic S
pyogenes is streptococcal sore throat or
pharyngitis. S pyogenes adhere to the pharyngeal
epithelium by means of surface pili.
 The illness may persist for weeks.
 May be characterized by intense nasopharyngitis,
tonsillitis, and intense redness and edema of the
mucous membranes, with purulent exudate

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 ERYSIPELAS

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 Streptococcal skin infections

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 Pharyngitis and tonsillitis

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3. Systemic infections
 Scarlet fever – strain of S. pyogenes carrying a
prophage that codes for pyrogenic toxin; can lead
to sequelae
 Septicemia
 Pneumonia
 Streptococcal toxic shock syndrome

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 Long-Term Complications of Group A Infections
Rheumatic fever - This is the most serious complication
because it results in damage to heart muscle and
valves.
 Certain strains of group A streptococci contain cell
membrane antigens that cross-react with human
heart tissue antigens.
 Rheumatic fever is often preceded by S pyogenes
infection 1–4 weeks earlier
Acute glomerulonephritis – This sometimes develops 3
weeks after S pyogenes skin infection
 AGN may be initiated by antigen-antibody complexes
on the
217 glomerular basement membrane. 03/11/2013 21:18
jimmy ongori 2013
Diagnostic Laboratory Tests
 Specimens - depend upon the nature of the infection. A
throat swab, pus, or blood for culture. Serum for antibody
tests.
 Smears - often show single cocci or pairs rather than
definite chains.
 Culture - on blood agar plates.
 Antigen Detection Tests - for rapid detection of group A
streptococcal antigen from throat swabs.
 Serologic Tests - A rise in the titer of antibodies to many
group A streptococcal antigens can be estimated. Such
antibodies include antistreptolysin O (ASO) – it is the most
widely used.
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Treatment
 Penicillin G, erythromycin.
 Antimicrobial drugs are also very useful in
preventing reinfection with -hemolytic group A
streptococci in rheumatic fever patients.
Prevention
(1) Detection and early antimicrobial therapy of
respiratory and skin infections with group A
streptococci.
(2) Antistreptococcal chemoprophylaxis in persons
who have suffered an attack of rheumatic fever.
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 α-Hemolytic Streptococci: Viridans Group
 Large complex group
 Streptococcus mutans, S. oralis, S. salivarus,
S. sanguis, S. milleri, S. mitis
 Most numerous and widespread residents of the
gums and teeth, oral cavity and also found in
nasopharynx, genital tract, skin
 Are not very invasive; dental or surgical procedures
facilitate entrance leading to; Bacteremia,
meningitis, abdominal infection, tooth abscesses

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 The most serious infection they cause is – subacute
endocarditis – blood-borne bacteria settle and grow
on heart lining or valves
 Persons with preexisting heart disease are at high risk.
 Colonization of heart by forming biofilms
 S. mutans produce slime layers that adhere to teeth - basis
for plaque. Involved in dental caries
 Persons with preexisting heart conditions should receive
prophylactic antibiotics before surgery or dental
procedures.

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 STREPTOCOCCUS PNEUMONIA
 The pneumococci (S pneumoniae) are gram-
positive diplococci, lancet-shaped or arranged in
chains.
 Causes 60-70% of all bacterial pneumonias
 Posses a capsule of polysaccharide
 Pneumococci are readily lysed by surface-active
agents
 Pneumococci are normal inhabitants of the upper
respiratory tract of 5–40% of humans and can
cause pneumonia, sinusitis, otitis, bronchitis,
bacteremia, meningitis, and other infectious
processes.
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 Epidemiology and Pathology
 5-50% of all people carry it as normal flora in the
nasopharynx; infections are usually endogenous.
 Very delicate, does not survive long outside its
habitat
 Young children, elderly, immune compromised,
those with other lung diseases or viral infections,
persons living in close quarters are predisposed to
pneumonia
 Pneumonia occurs when cells are aspirated into the
lungs of susceptible individuals.
 Gains access to middle ear by way of Eustachian
tube

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PATHOGENICITY
 Cause lobar pneumonia, bronchitis,meningitis,
bacteremia, otitis media, sinusitis, conjuctivitis
 Severe infection in the elderly, immune compromised,
splenectomy patients
 Pneumonia in children especially in sickle cell disease
 Are normal flora in the upper respiratory tract
 Pneumococci produce disease through their ability to
multiply in the tissues.
 They produce no toxins of significance.
 The virulence of the organism is due to capsule, which
prevents or delays ingestion by phagocytes.
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Diagnostic Laboratory Tests

 Blood, CSF and sputum - smear and culture.

 Sputum;
 Stained Smears - A Gram-stained film shows
typical organisms, many polymorphonuclear
neutrophils, and many red cells.
 Culture - The culture is created by sputum
cultured on blood agar and incubated in CO2 or
a candle jar. A blood culture is also taken.

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Treatment
 Since pneumococci are sensitive to many
antimicrobial drugs, early treatment usually results
in rapid recovery
 Penicillin G is effective in treating pneumonia
caused by pneumococci but would not be effective
in treatment of meningitis due to the same strains.
 Some penicillin-resistant strains are resistant to
cefotaxime. Resistance to tetracycline and
erythromycin occurs also.
 Pneumococci remain susceptible to vancomycin.
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Prevention, & Control
 Pneumococcal pneumonia accounts for about 60%
of all bacterial pneumonias.
 Predisposing factors are more important than
exposure to the infectious agent, and the healthy
carrier is more important in spreading pneumococci
than the sick patient.
 Vaccines can provide upto 90% protection
 Vaccine is appropriate for elderly, debilitated, or
immunosuppressed individuals.
 A pneumococcal conjugate vaccine is currently being
given to children <1yr in Kenya
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 Group B: Streptococcus agalactiae
 Regularly resides in human vagina, pharynx and
large intestine
 Can be transferred to infant during delivery and
cause severe infection
 most prevalent cause of neonatal pneumonia,
sepsis, and meningitis
 Pregnant women should be screened and treated.
 Wound and skin infections and endocarditis in
debilitated people

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 NEISSERIA

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 Family Neisseriaceae
 Gram-negative cocci
 Residents of mucous membranes of warm-blooded
animals
 Genera include Neisseria, Moraxella, Acinetobacter.
 2 primary human pathogens:
 Neisseria gonorrhoeae
 Neisseria meningitidis

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 Genus Neisseria
 Gram-negative, bean-shaped, diplococci
 None develop flagella or spores.
 Capsules on pathogens
 Pili
 Strict parasites, do not survive long outside of the
host
 Aerobic or microaerophilic
 Produce catalase and cytochrome oxidase
 Pathogenic species require enriched complex media
and CO2.
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Neisseria gonorrhoeae: The Gonococcus
 The typical Neisseria is a gram-negative, nonmotile
diplococcus
 Individual cocci are kidney-shaped; when the
organisms occur in pairs, the flat or concave sides
are adjacent.
 Found only in man
 Is the causative agent of gonorrhea, the second
most common venereal disease.
 Virulence factors: pili, other surface molecules for
attachment - slows phagocytosis
 IgA protease – cleaves secretory IgG
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•These bacteria grow best on chocolate agar (so-
called because it contains heated blood, brown in
color); a modified (selective) chocolate agar
commonly used is Thayer Martin.

•The colonies are oxidase positive (i.e. produce

cytochrome oxidase) which is demonstrated by
flooding the plate with a dye which on oxidation
changes color.

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 Epidemiology of Gonorrhea
Seriously underreported STD
Found only in humans with strikingly different
epidemiological presentations for females and
males
Asymptomatic carriage is a major reservoir
Transmission primarily by sexual contact
Lack of protective immunity and therefore
reinfection, partly due to antigenic diversity of
strains
Does not survive more than 1-2 hours on fomites

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 Pathogenesis of Neisseria gonorrhoeae
 Fimbriated cells attach to intact mucus membrane
epithelium
 Capacity to invade intact mucus membranes or
skin with abrasions
 Adherence to mucosal epithelium
 Penetration into and multiplication before
passing through mucosal epithelial cells
 Establish infection in the sub-epithelial layer
 Most common sites of inoculation:
 Cervix (cervicitis) or vagina in the female
jimmy
Urethra
ongori 2013
(urethritis) or penis in the male
03/11/2013 21:18
235
 Attack mucous membranes of the GUT, eye,
rectum, and throat, producing acute suppuration
that may lead to tissue invasion; this is followed
by chronic inflammation and fibrosis.
 In males - urethritis, with yellow, creamy pus and
painful urination. May extend to the epididymis.
 As suppuration subsides in untreated infection,
fibrosis occurs, sometimes leading to urethral
strictures
 Urethral infection in men can be asymptomatic.

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 In females, the 10 infection is in the endocervix and
extends to the urethra and vagina, giving rise to
mucopurulent discharge. May progress to the uterine
tubes, causing salpingitis, fibrosis, and obliteration of
the tubes.
 Infertility occurs in 20% of women with gonococcal
salpingitis.
 Chronic gonococcal cervicitis or proctitis is often
asymptomatic.
 Gonococcal bacteremia leads to skin lesions on the
hands, forearms, feet, and legs and to tenosynovitis and
suppurative arthritis, usually of the knees, ankles, and
wrists.
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 Gonococcal endocarditis is an uncommon but
severe infection.
 Gonococci sometimes cause meningitis and eye
infections in adults
 Gonococcal ophthalmia neonatorum, an infection
of the eye of the newborn, is acquired during
passage through an infected birth canal.
 To prevent gonococcal ophthalmia neonatorum,
instillation of tetracycline, erythromycin, or silver
nitrate into the conjunctival sac of the newborn is
compulsory in the United States.
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 GONORRHEA IN MEN:
Urethritis; Epididymitis
• Most infections among men are acute and
symptomatic with purulent discharge & dysuria
after 2-5 day incubation period
• Male host seeks treatment early preventing serious
sequelae
• The two bacterial agents primarily responsible for
urethritis among men are N. gonorrhoeae and
Chlamydia trachomatis

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 GONORRHEA IN WOMEN
Cervicitis; Vaginitis; Pelvic Inflammatory Disease
(PID); Disseminated Gonococcal Infection (DGI)
Women are often asymptomatic or have atypical
indications (unrecognized S/S)
Often untreated until PID complications develop
 Pelvic Inflammatory Disease (PID)
• May also be asymptomatic, but difficult diagnosis
accounts for many false negatives
• Can cause scarring of fallopian tubes leading to
infertility or ectopic pregnancy

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 IN WOMEN (cont.) :
Disseminated Gonococcal Infection (DGI):
• Result of gonococcal bacteremia
• Skin lesions
• Petechiae (small, purplish, hemorrhagic spots)
• Pustules on extremities
• Arthralgias (pain in joints)
• Tenosynovitis (inflammation of tendon sheath)
• Septic arthritis
• Occasional complications: Hepatitis; Rarely
endocarditis or meningitis

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 242

Females Males
50% risk of infection after single exposure 20% risk of infection after single
exposure
Asymptomatic infections frequently not Most initially symptomatic (95% acute)
diagnosed
Major reservoir is asymptomatic carriage Major reservoir is asymptomatic
in females carriage in females
Genital infection primary site is cervix Genital infection generally restricted to
(cervicitis), but vagina, urethra, rectum urethra (urethritis) with purulent
can be colonized discharge and dysuria
Ascending infections in 10-20% including Rare complications may include
salpingitis, tubo-ovarian abscesses, pelvic epididymitis, prostatitis, and
inflammatory disease (PID) , chronic periurethral abscesses
infections can lead to sterility
Disseminated infections more common, Disseminated infections are very rare
including septicemia, infection of skin and
joints (1-3%)
Can infect infant
jimmy ongori at
2013delivery (conjunctivitis, More common in homosexual/bisexual
opthalmia neonatorum) men than in heterosexual populatiuon
243 jimmy ongori 2013 03/11/2013 21:18
244 jimmy ongori 2013 03/11/2013 21:18
Diagnostic Laboratory Tests
 Specimens - Pus and secretions are taken from the urethra,
cervix, rectum, conjunctiva, throat, or synovial fluid for
culture and smear
 Smears - Gram-stained smears - diplococci within pus cells.
 Culture on enriched selective medium eg, modified Thayer-
Martin medium at 37 °C.
 Oxidase test is positive
 Nucleic Acid Amplification Tests - nucleic acid amplification
assays are available for direct detection of N gonorrhoeae in
genitourinary specimens.
 Serology - by immunoblotting, radioimmunoassay, and
ELISA (enzyme-linked immunosorbent assay) tests
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 Prevention & Treatment
• Penicillin no longer drug of choice due to resistance
• Uncomplicated infection: ceftriaxone, cefixime or
Fluoroquinolone. Can be combined with doxycycline or
azithromycin for dual infections with Chlamydia
• Chemoprophylaxis of newborns against opthalmia
neonatorum with 1% silver nitrate, 1% tetracycline, or 0.5%
erythromycin eye ointments
• Treatment of newborns with opthalmia neonatorum with
ceftriaxone
• Measures to limit epidemic include education, aggressive
detection, and follow-up screening of sexual partners, use of
condoms
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 Neisseria meningitides
 Aerobic, non-spore-forming, Gram-negative
diplococci
 Encapsulated (capsule the major virulence factor)
 Cytochrome oxidase positive
 Natural habitat - nasopharyngeal tract of humans .
 5-15% of the human population carries the bacteria
in its nonpathogenic form .
 Also known as meningococcus
 Only infects humans, there is no animal reservoir.

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 Oxidase +ve
 Acid production from glucose and
maltose, but not sucrose or lactose
 Neisseria gonorrhoeae, N. meningitidis, and
Moraxella catarrhalis are capnophilic (optimal
growth with 3-7% CO2)
 Growth of Neisseria meningitidis occurs on
both sheep blood and chocolate agar

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 Pathogenicity
• Pili-mediated, receptor-specific colonization of
nonciliated cells of nasopharynx
• Antiphagocytic polysaccharide capsule allows
systemic spread in absence of specific immunity
• Toxic effects mediated by hyperproduction of
lipooligosaccharide
• Second most common cause (behind S.
pneumoniae) of community-acquired meningitis
in previously healthy adults; swift progression
from good health to life-threatening disease
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Diseases Associated with N. meningitidis
Following dissemination of virulent organisms from
the nasopharynx:
• Meningitis
• Septicemia (meningococcemia) with or
without meningitis
• Meningoencephalitis
• Pneumonia
• Arthritis
• Urethritis
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 Epidemiology of Meningococcal Disease
• Humans only natural hosts
• Person-to-person transmission by aerosolization of
respiratory tract secretions in crowded conditions
• Close contact with infectious person
• Highest incidence in children younger than 5 years
and particularly those younger than 1 year of age as
passive maternal antibody declines
• Commonly colonize nasopharynx of healthy
individuals; highest oral and nasopharyngeal
carriage rates in school-age children, young adults
and lower socioeconomic groups
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 VIRULENCE FACTORS
•Lipooligosaccharide (LOS) is a component of the
outer membrane of N. meningitidis which acts as an
endotoxin which is responsible for fever, septic shock,
and hemorrhage due to the destruction of red blood
cells.
• A polysaccharide capsule which prevents
phagocytosis and aids in evasion of the host immune
response
•Fimbriae which mediate attachment of the
bacterium to the epithelial cells of the nasopharynx

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 Pathogenesis of Meningococcal Disease
Specific receptors for bacterial fimbriae on nonciliated
columnar epithelial cells in nasopharynx of host
Organisms are internalized into phagocytic
vacuoles, avoid intracellular killing in absence of
humoral immunity and complement system
Replicate intracellularly
Hyperproduction of endotoxin (lipid A of LOS)
mediates most clinical manifestations including diffuse
vascular damage (e.g., endothelial damage, vasculitis
(inflammation of vessel walls), thrombosis (clotting), 253
disseminated
jimmy ongori 2013 intravascular coagulation (DIC) 03/11/2013 21:18
 Laboratory Diagnosis
• Large numbers of encapsulated, small, gram-negative
diplococci and polymorphonuclear leukocytes
(PMN’s) can be seen microscopically in
cerebrospinal fluid
• Transparent, non-pigmented nonhemolytic colonies on
chocolate blood agar with enhanced growth in
moist atmosphere with 5% CO2
• Oxidase-positive
• Acid production from glucose and maltose but not
from other sugars
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Laboratory Diagnosis
 Specimens – blood, CSF, Nasopharyngeal swab, Puncture
material from petechiae may be taken for smear and culture.
 Smears - Gram-stained smears show typical neisseriae within
polymorphonuclear leukocytes or extracellularly.
 Culture – CSF on "chocolate" agar at 37 °C in of 5% CO2
(candle jar).
 A modified Thayer-Martin medium with antibiotics
(vancomycin, colistin, amphotericin) favors the growth of
neisseriae, inhibits many other bacteria, and is used for
nasopharyngeal cultures.
 Serology - Antibodies to meningococcal polysaccharides can
be measured by latex agglutination or hemagglutination tests
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 Prevention and Treatment
• Penicillin is drug of choice
• Chloramphenicol or cephalosporins as alternatives
• Chemoprophylaxis of close contacts with rifampicin
or sulfadiazine (if susceptible)
• Reduction of personal contacts in a population with
a high carrier rate. This is accomplished by
avoidance of crowding.
• Polyvalent vaccine in people older than 2 years of
age for immunoprophylaxis as an adjunct to
chemoprophylaxis
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 BACILLI

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 Medically Important Gram-Positive Bacilli
Three general groups:
1. Endospore-formers
Bacillus, Clostridium
2. Non-endospore-formers
Listeria, Erysipelothrix
3. Irregular shaped and staining properties
Corynebacterium, Proprionibacterium,
Mycobacterium, Actinomyces, Nocardia

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259 jimmy ongori 2013 03/11/2013 21:18
 Spore-forming Gram +VE Bacilli
 Genus Bacillus
 Genus Clostridium

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General Characteristics of the Genus Bacillus
 Gram +VE, endospore-forming, motile rods
 Mostly saprophytic, prevalent in soil, water, and air
and on vegetation. B. cereus can grow in foods and
produce an enterotoxin or an emetic toxin and
cause food poisoning
 Aerobic and catalase positive
 Versatile in degrading complex macromolecules
 Primary habitat is soil
 2 species of medical importance:
 Bacillus anthracis
 Bacillus cereus
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jimmy ongori 2013
 Bacillus anthracis
 Large, block-shaped rods
 Gram +VE rod
 Facultative anaerobe
 Central spores that develop under all
conditions except in the living body
 Virulence factors – polypeptide capsule and
exotoxins

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 Endospore
 Oxygen required for sporulation
 1 spore per cell
 Dehydrated cells
 Highly resistant to heat, cold, chemicals,
disinfectants, dry periods
 Protoplast carries the material for future vegetative
cell
 Cortex provides heat and radiation resistance
 Spore wall provides protection from chemicals &
enzymes
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264 jimmy ongori 2013 03/11/2013 21:18
 Epidemiology of Bacillus anthracis
• Anthrax is primarily a disease of herbivores—goats,
sheep, cattle, horses, etc
• Enzootic in certain countries (e.g., Turkey, Iran,
Pakistan,and Sudan)
• Anthrax spores are infectious for decades
• Used Biologic warfare experiments - bioterrorism
•Three well-defined cycles
• Survival of spores in the soil
• Animal infection
• Infection in humans

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jimmy ongori 2013
 Epidemiology of Bacillus anthracis (cont.)
 Primarily a disease of herbivorous animals
 Most commonly transmitted to humans by direct
contact with animal products (e.g., wool and hair)
 Also acquired via inhalation & ingestion
• Increased mortality with these portals of entry
 Still poses a threat
• Importing materials contaminated with spores
(e.g., bones, hides, and other materials)
• Usually encountered as an occupational disease
among Veterinarians, agricultural workers

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jimmy ongori 2013
 Clinical Presentation of Anthrax
1. Cutaneous Anthrax
• 95% human cases are cutaneous infections
• 1 to 5 days after contact
• Small, pruritic, non-painful papule at inoculation
site, Papule develops into hemorrhagic vesicle &
ruptures
• Slow-healing painless ulcer covered with black
eschar surrounded by edema
• Infection may spread to lymphatics
• Septicemia may develop
• 20% mortality in untreated cutaneous anthrax
267 jimmy ongori 2013 03/11/2013 21:18
268 jimmy ongori 2013 03/11/2013 21:18
 2. Inhalation Anthrax
• Virtually 100% fatal (pneumonic)
• Meningitis may complicate cutaneous and
inhalation forms of disease
• Pharyngeal anthrax
• Fever
• Pharyngitis
• Neck swelling
3. Gastrointestinal (Ingestion) Anthrax
• Virtually 100% fatal
• Abdominal pain
• Hemorrhagic ascites
• Paracentesis
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fluid may reveal gram-positive rods
03/11/2013 21:18
269
 Laboratory Diagnosis
 Gram stain
 Culture of B. anthracis from the blood, skin lesions,
vesicular fluid, or respiratory secretions
 Rapid detection methods
- PCR for detection of nucleic acid
- ELISA assay for antigen detection
- Other immunohistochemical and
immunoflourescence examinations

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 Treatment and Prevention
• Penicillin is drug of choice
• Ciprofloxacin, Erythromycin, chloramphenicol
• Doxycycline now commonly recognized as prophylactic
• Soil is contaminated with anthrax spores from the
carcasses of dead animals. These spores remain viable
for decades. Control measures include
• Disposal of animal carcasses - burning or by deep
burial in lime pits
• Decontamination of animal products
• Protective clothing and gloves for handling potentially
infected materials
• Immunization of domestic animals and Persons with
high occupational risk.
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Bacillus cereus
 Gram +ve bacilli
 Spore forming
 Facultative anaerobe
 Non-fastidious growth requirements
Virulence
 Enterotoxin
 Spores can survive in soil
 Tissue destruction mediated by cytotoxic enzymes –
cereolysin and phospholipase C
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Epidemiology
 Ubiquitous in soils throughout the world
 People at risk include those who consume food
contaminated with the bacterium (eg rice, meat,
vegetables, sauces), those with penetrating injuries
and those who receive intravenous injections
Diseases
 Infections include emetic (vomiting) and diarrheal
forms of GE, ocular infection after trauma to the eye
and other opportunistic infections

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Diagnosis
 Isolation of the organism in implicated food product
or nonfecal specimen (eg eye, wound)

Treatment, Prevention, and Control

 GIT infections treated symptomatically
 Ocular or other invasive diseases – vancomycin,
clindamycin, ciprofloxacin, or gentamicin
 GIT disease prevented by proper preparation of food

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 CLOSTRIDIA 275

 Large Gram +VE, Straight or slightly curved rods with

slightly rounded ends
 Anaerobic
 Spore bearing
 Saprophytes
 Some are commensals of the animal & human gut
which invade the blood and tissue when host die
and initiate the decomposition of the corpse
 Causes diseases such as gas gangrene, tetanus,
botulism & pseudo-membranous colitis by
producing toxins which attack the neurons03/11/2013
pathways 21:18
jimmy ongori 2013
 Clostridia of medical importance
Clostridium
Causing

Tetanus Botulism ِAntibiotic associated diarrhea

Gas gangrene e.g. Cl. difficille
e.g. Cl. tetani e.g. Cl. botulinum

Saccharolytic Proteolytic
e.g. Cl. perfringens &Cl. septicum e.g. Cl. sporogenes

Mixed: Cl. histolyticum

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 CLOSTRIDIUM CAUSING TETANUS
Clostridium tetani
 Gram +ve, straight, slender rod with rounded ends
 All species form endospores (drumstick with a large
round end)
 Fermentative
 Obligate anaerobe
 Motile by peritrichous flagella
 Grows well in cooked meat broth and produces a
thin spreading film when grown on enriched blood
agar
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 Spores are highly resistant to adverse conditions
 Spores do not germinate and growth does not
normally proceed unless a suitably low redox
potential exists
 Iodine (1%) in water is able to kill the spores within
a few hours

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 Toxins
Cl. tetani produces two types of toxins:
 Tetanolysin, which causes lysis of RBCs
 Tetanospasmin is a neurotoxin and essential
pathogenic product
 Tetanospasmin is toxic to humans and various
animals when injected parenterally, but it is not toxic
by the oral route
 Tetanospasmin causes increasing excitability of spinal
cord neurons and muscle spasm
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Pathogenesis
 C tetani is not an invasive organism - Infection
remains strictly localized in the area of devitalized
tissue (wound, burn, injury, umbilical stump, surgical
suture) into which the spores have been introduced.
 Germination of the spore and development of
vegetative organisms that produce toxin are aided
by (1) necrotic tissue, (2) calcium salts, and (3)
associated pyogenic infections, all of which aid
establishment of low oxidation-reduction potential.
 The toxin released from vegetative cells reaches the
central nervous system and rapidly becomes fixed to
receptors in the spinal cord and brain stem03/11/2013 21:18
280
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Clinical Findings
 Incubation period 4–5 days to weeks.
 Characterized by tonic contraction of voluntary muscles.
Muscular spasms often involve first the area of injury and infection
and then the muscles of the jaw (trismus, lockjaw), which contract
so that the mouth cannot be opened.
 Gradually, other voluntary muscles become involved, resulting in
tonic spasms. Any external stimulus may precipitate a tetanic
generalized muscle spasm.
 The patient is fully conscious, and pain may be intense. Death
usually results from interference with the mechanics of
respiration.
 The mortality rate in generalized tetanus is very high.
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 Laboratory Diagnosis of Tetanus
 The diagnosis of tetanus depends primarily upon the clinical
manifestation of tetanus including muscle spasm and rigidity.
 Specimen: Wound exudates using capillary tube
 Culture:
 On blood agar and incubated anaerobically
 Growth appears as a fine spreading film.
 Gram stain
 Cl. tetani is Gram positive rod motile with a round
terminal spore giving a drumstick appearance

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Prevention & Treatment
 Muscle relaxants, sedation, and assisted ventilation for
patients with symptoms
 Prevention is most important and depends upon;
 active immunization with toxoids - TT
 proper care of wounds contaminated with soil, etc
 prophylactic use of antitoxin
 administration of penicillin.
 Human antitoxin (tetanus immune globulin) gives
adequate systemic protection - It neutralizes the toxin
that has not been fixed to nervous tissue.

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 Surgical debridement is vitally important because it
removes the necrotic tissue that is essential for
proliferation of the organisms.
 Penicillin strongly inhibits the growth of C tetani and
stops further toxin production.
 Antibiotics may also control associated pyogenic
infection.
 When a previously immunized individual sustains a
potentially dangerous wound, an additional dose of
toxoid should be injected to restimulate antitoxin
production.
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Control
 Tetanus is a totally preventable disease.
 Immunization with tetanus toxoid
 Tetanus toxoid is produced by detoxifying the toxin with
formalin and then concentrating it.
 In young children, tetanus toxoid is often combined with
diphtheria toxoid, pertussis, Hepatitis B and Haemophilus
influenza vaccine (Pentavalent) and given at 6 weeks, 10 weeks
and 14 weeks of life, previously given as DPT
 Control measures are not possible because of the wide
dissemination of the organism in the soil and the long survival
of its spores.
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 Clostridium Causing Gas Gangrene
Clostridia causing gas gangrene

Saccharolytic organisms Proteolytic organisms

Cl. perfringens, Cl. septicum Cl. sporogenes
Ferment carbohydrates Digest proteins with blackening bad smell
Acid and gas are produced production

Mixed saccharolytic & proteolytic

Cl. histolyticum

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 Saccharolytic Microorganisms

Cl. perfringens
Causing

Food poisoning
Gas gangrene
(Enterotoxin)

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 Clostridium perfringens
 Large Gram-positive bacilli with stubby ends
 Capsulated
 Non motile (Cl. tetani is motile)
 Anaerobic
 Grown quickly on selective media
 Can be identified by Nagler reaction
 Clostridium perfringens most frequent clostridia involved in
soft tissue and wound infections - myonecrosis
 Spores found in soil, human skin, intestine, and vagina
 Predisposing factors – surgical incisions, compound
fractures, diabetic ulcers, septic abortions, puncture
wounds, gunshot wounds
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 Toxins
  toxin (phospholipase C, lecithinase) is the most
important toxin
 Lyses of RBCs, platelets, leucocytes and
endothelial cells
 Increased vascular permeability with massive
hemolysis and bleeding, tissue destruction
 Hepatic toxicity and myocardial dysfunction
 -toxin is responsible for necrotic lesions in
necrotizing enterocolitis
 Enterotoxin is heat labile toxin produced in colon →
food poisoning 03/11/2013 21:19
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 Pathology
 Not highly invasive; requires damaged and dead
tissue and anaerobic conditions
 Conditions stimulate spore germination, vegetative
growth and release of exotoxins, and other virulence
factors.
 Fermentation of muscle carbohydrates results in the
formation of gas and further destruction of tissue.

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Clinical Diseases
1.Soft tissue infections - Portal of entry: trauma or
intestinal tract.
•Usually caused by mixed infection including toxigenic
clostridia, proteolytic clostridia and various cocci and
gram-negative organisms.Three types of infections:
 Cellulitis: gas formation in the soft tissue.
Fasciitis or suppurative myositis: accumulation of
gas in the muscle planes.
Myonecrosis or gas gangrene: a life-threatening
disease.
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2. Gas gangrene
•Spores germinate, vegetative cells multiply, ferment
carbohydrates and produce gas in the tissue. This
results in distension of tissue and interference with
blood supply
•Bacteria produce necrotizing toxin and hyaluronidase,
which favor the spread of infection, tissue necrosis
extends, resulting in increased bacterial growth,
hemolytic anemia, then severe toxemia and death.
Incubation: 1-7 days after infection.
Can be also caused by other Clostridium species.

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3. Food poisoning
The enterotoxin causes marked hypersecretion in
jejunum and ileum.
Enterotoxin: a heat-labile protein produced by some
strains of C. perfringens type A. When >108 cells in
contaminated meat are ingested and sporulate in the
small intestine, enterotoxin is formed.
•It disrupts ion transport in the enterocytes
•Symptoms: diarrhea, usually without vomiting or
fever.
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 Laboratory Diagnosis
Specimen: Histological specimen/wound exudates
Microscopical examination (Gram, Spore stain
etc)
Gram-positive bacilli, non motile, capsulated &
sporulated
The spore is oval, sub-terminal & non bulging
Spores are rarely observed
Culture: Anaerobically at 370C
On Robertson's cooked meat medium →
blackening of meat will observed with the production of
H2S and NH3
On blood agar → β-hemolytic colonies 03/11/2013 21:19
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 Treatment and Prevention
 Immediate cleansing of dirty wounds, deep
wounds, decubitus ulcers, compound fractures,
and infected incisions
 Debridement of disease tissue
 Large doses of cephalosporin or penicillin
 Hyperbaric oxygen therapy
 No vaccines available

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 Clostridium difficile-Associated Disease
 Normal resident of colon, in low numbers
 Causes antibiotic-associated colitis
 relatively non-invasive; treatment with broad-
spectrum antibiotics kills the other bacteria,
allowing C. difficile to overgrow
 Produces enterotoxins that damage intestines
 Major cause of diarrhea in hospitals
 Increasingly more common in community acquired
diarrhea

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 Treatment and Prevention
 Mild uncomplicated cases respond to fluid and
electrolyte replacement and withdrawal of
antimicrobials.
 Severe infections treated with oral vancomycin
or metronidazole and replacement cultures
 Increased precautions to prevent spread

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 Clostridial Food Poisoning
 Clostridium botulinum – rare but severe
intoxication usually from home canned food
 Clostridium perfringens – mild intestinal
illness; second most common form of food
poisoning worldwide

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 BOTULINUM FOOD POISONING - BOTULISM
 Botulism – intoxication associated with inadequate
food preservation
Clostridium botulinum
 Gram positive, Spore-forming, Obligate anaerobic
bacillus
 Spores
 Ubiquitous, Resistant to heat, light, drying and
radiation
 Commonly inhabits soil and water

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 Neurotoxins
 Seven different types: A - G
 All cause flaccid paralysis
 Only a few nanograms can cause illness
 Binds neuromuscular junctions
 Toxin: Destroyed by boiling
 Spores: Higher temperatures to be inactivated

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 Pathogenesis
 Spores are present on food when gathered and
processed.
 Anaerobic conditions favor spore germination and
vegetative growth.
 Potent toxin, botulin, is released.
 Toxin is carried to neuromuscular junctions and
blocks the release of acetylcholine, necessary for
muscle contraction to occur.
 Double or blurred vision, difficulty swallowing,
neuromuscular symptoms
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Transmission
 Ingestion of – Organism, Spores, Neurotoxin
 Wound contamination
 Inhalation
Human Disease
 Three forms
 Foodborne
 Wound
 Infant
 All forms fatal and a medical emergency
 Incubation period: 12-36 hours

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1.Foodborne Botulism
 Preformed toxin ingested from contaminated food
 Most common from home-canned foods
 Asparagus, green beans, beets, corn, baked
potatoes, garlic, chile peppers, tomatoes
2.Infant Botulism
 Spore ingestion - Germinate then toxin released and
colonize large intestine
 Infants < 1 year old, but mostly < 6 months old
 Spores from varied sources - Honey, food, dust, corn
syrup
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 3. Wound Botulism
 Organism enters wound
 Develops under anaerobic conditions
 From ground-in dirt or gravel
 It does not penetrate intact skin

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 Prevention
 Do not feed honey to children <1 yr of age
 Proper food preservation methods
 Proper time, temperature and pressure
 80oC for 30 min or 100oC for 10 min
 Prompt refrigeration of foods
 Boil foods for > 10 minutes
 Decontamination
 Boil suspected food before discarding
 Boil or chlorine disinfect utensils used
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 Clostridial Gastroenteritis
 Clostrium perfringens
 Spores contaminate food that has not been
cooked thoroughly enough to destroy spores.
 Spores germinate and multiply (especially if
unrefrigerated).
 When consumed, toxin is produced in the
intestine; acts on epithelial cells, acute
abdominal pain, diarrhea, and nausea
 Rapid recovery

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Gram-Positive Non-Spore-Forming Bacilli

Medically important genera:

 Corynebacterium
 Proprionibacterium
 Mycobacterium
 Actinomyces
 Nocardia
 Listeria monocytogenes
 Erysipelothrix rhusiopathiae
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 CORYNEBACTERIA (Genus Corynebacterium)
Gram positive bacilli
Aerobic or facultatively anaerobic
Small, pleomorphic (club-shaped), gram-positive
bacilli that appear in short chains (“V” or “Y”
configurations) or in clumps resembling “Chinese
letters”
Cells contain metachromatic granules
Lipid-rich cell wall contains meso-diaminopimelic
acid, arabino-galactan polymers, and short-chain
mycolic acids

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 Pathogenic Corynebacterial Species

Corynebacterium diphtheriae
Corynebacterium jeikeium
Corynebacterium urealyticum

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Corynebacterium diphtheriae
 Aerobic, Gram positive, Noncapsulated, rods
 Gray-black colonies on medium
 Metachromatic granules
 Reservoir of healthy carriers; potential for
diphtheria is always present
 Most cases occur in non-immunized children living
in crowded, unsanitary conditions.
 Acquired via respiratory droplets from carriers or
actively infected individuals

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Transmission
 Solely among humans
 Spread by droplets
 Secretions
 Direct contact

Risk factors
 Poor nutrition
 Crowded or unsanitary living conditions
 Low vaccine coverage among infants and children
 Immunity gaps in adults
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 Diphtheria toxin
 Is a heat-labile polypeptide that can be lethal
in a dose of 0.1 g/kg.
 It is assumed that the abrupt arrest of protein
synthesis is responsible for the necrotizing and
neurotoxic effects of diphtheria toxin.
 Toxin diffuses throughout body via blood
- Cardiac, neurologic complications
- Heart/respiratory damage, paralysis

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Pathology 313

 Diphtheria toxin is absorbed into the mucous

membranes and causes destruction of epithelium
and a superficial inflammatory response.
 A grayish "pseudomembrane" is formed—
commonly over the tonsils, pharynx, or larynx.
 The regional lymph nodes in the neck enlarge, and
there may be marked edema of the entire neck.
 The diphtheria bacilli within the membrane
continue to produce toxin actively. This is absorbed
and results in distant toxic damage
 Nerve damage, resulting often in paralysis of the
jimmy ongori 2013
soft palate, eye muscles, or extremities. 03/11/2013 21:19
Diagnostic Laboratory Tests
 Dacron swabs from the nose, throat, or other lesions.
Swabs should be collected from beneath any visible
membrane.
 Smears stained with alkaline methylene blue or Gram stain
show beaded rods in typical arrangement.
 Loeffler slant may yield organisms of typical "diphtheria-
like" morphology. In 36–48 hours. Colonies on tellurite
medium are sufficiently definite for recognition of C
diphtheriae.
 Toxigenicity tests
 PCR,jimmyELISA
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 Control
 Sanitary: Reduce carrier rate by use of vaccine.
 Immunological: A vaccine (Pentavalent) prepared
from an alkaline formaldehyde inactivated toxin (i.e.
toxoid) is required.
 Passive immunization with antitoxin can be used for
patients.
 Chemotherapeutic: Penicillin, erythromycin or
gentamicin are drugs of choice.

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 Mycobacteria: Acid-Fast Bacilli

 Mycobacterium tuberculosis
 M. leprae
 M. avium complex
 M. fortuitum
 M. marinum
 M. scrofulaceum
 M. paratuberculosis

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 Genus Mycobacterium
 Gram-positive irregular bacilli
 Acid-fast staining
 Strict aerobes
 Produce catalase
 Possess mycolic acids and a unique type of
peptidoglycan
 Do not form capsules, flagella or spores
 Grow slowly

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 Mycobacterium tuberculosis
 Tubercle bacillus
 Once stained they resist decolorization by acid or alcohol and
are therefore called "acid-fast" bacilli.
 Mycobacteria cannot be classified as either gram-positive or
gram-negative. Once stained by basic dyes they cannot be
decolorized by alcohol, regardless of treatment with iodine.
 Produces no exotoxins or enzymes that contribute to
infectiousness
 Virulence factors - contain complex waxes and cord factor
that prevent destruction by lysosomes or macrophages
 Mycobacterium tuberculosis is the etiologic agent of
tuberculosis in humans. Humans are the only reservoir for
the bacterium.
 Chains of cells in smears made from in vitro-grown colonies
often form distinctive serpentine cords
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 Cell Wall Structure
 The cell wall structure is unique among prokaryotes, and it is a
major determinant of virulence for the bacterium.
 The cell wall complex contains peptidoglycan, but otherwise it
is composed of complex lipids. Over 60% of the mycobacterial cell
wall is lipid. The lipid fraction of MTB's cell wall consists of three
major components, mycolic acids, cord factor, and wax-D.
i. Mycolic acids are unique alpha-branched lipids found in cell
walls of Mycobacterium and Corynebacterium. They make up 50% of
the dry weight of the mycobacterial cell envelope. Mycolic acids
are strong hydrophobic molecules that form a lipid shell around the
organism and affect permeability properties at the cell
surface. Mycolic Acids are thought to be a significant determinant
of virulence in MTB.
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 ii. Cord Factor is responsible for the serpentine
cording mentioned above. Cord factor is toxic to
mammalian cells and is also an inhibitor of PMN
migration. Cord factor is most abundantly
produced in virulent strains of MTB.
iii.Wax-D in the cell envelope is the major
component of Freund's complete adjuvant
(CFA).

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The high concentration of lipids in the cell wall of
Mycobacterium tuberculosis have been associated
with these properties of the bacterium:

 Impermeability to stains and dyes

 Resistance to many antibiotics
 Resistance to killing by acidic and alkaline compounds
 Resistance to osmotic lysis via complement
deposition
 Resistance to lethal oxidations and survival inside of
macrophages .
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 Epidemiology
 M. tuberculosis infects one third world’s population
 Causes 8 million new cases active disease annually, Causes
2 million deaths - 2nd only to HIV as cause of death from
infectious agent world wide among adults
 HIV/TB relationship has exacerbated problem with TB
increasing in areas with high AIDS incidence- Especially
sub-Saharan Africa
 Kenya has a large and rising TB disease burden and is
ranked among the twenty-two countries that collectively
contribute about 80% of the world's TB cases.
 There were 106,083 in 2010
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 Epidemiology Cont’
 The most frequent source of infection is the human who
excretes, particularly from the respiratory tract, large
numbers of tubercle bacilli.
 Risk factors;
Exposure to sources of infectious bacilli—principally
sputum-positive patients, Rate of active infection in the
population, Crowding, Low socioeconomic status,
Inadequacy of medical care, Age (high risk in infancy
and in the elderly), Undernutrition, Immunologic
status, coexisting diseases (eg, silicosis, diabetes), and
other individual host resistance factors.
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 Pathogenesis
 Mycobacteria in droplets 1–5 m in diameter are inhaled
and reach alveoli. The disease results from establishment
and proliferation of virulent organisms and interactions
with the host.
 Resistance and hypersensitivity of the host greatly
influence the development of the disease.
Pathology
 The production and development of lesions and their
healing or progression are determined by; (1) the number
of mycobacteria in the and (2) the resistance and
hypersensitivity of the host. Two Principal Lesions;
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i. Exudative Type - consists of an acute inflammatory reaction,
with edema fluid, PMN leukocytes, and, later, monocytes around
the tubercle bacilli. It may heal by resolution, so that the entire
exudate becomes absorbed; it may lead to massive necrosis of
tissue; or it may develop into the second (productive) type of
lesion.
ii. Productive Type - When fully developed, this lesion, a chronic
granuloma, consists of three zones: (1) a central area of large,
multinucleated giant cells containing tubercle bacilli; (2) a mid
zone of pale epithelioid cells, and (3) a peripheral zone of
fibroblasts, lymphocytes, and monocytes.
• Later, peripheral fibrous tissue develops, and the central area
undergoes caseation necrosis. Such a lesion is called a tubercle. A
caseous tubercle may break into a bronchus, empty its contents
there, and form a cavity. It may subsequently heal by fibrosis or
calcification.
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 Spread of Organisms in the Host
 Spread by direct extension, through the lymphatic channels and
bloodstream, and via the bronchi and gastrointestinal tract.
 In the first infection, tubercle bacilli always spread from the initial
site via the lymphatics to the regional lymph nodes.
 The bacilli may spread farther and reach the bloodstream, which in
turn distributes bacilli to all organs (miliary distribution). The
bloodstream can be invaded also by erosion of a vein by a caseating
tubercle or lymph node. If a caseating lesion discharges its contents
into a bronchus, they are aspirated and distributed to other parts of
the lungs or are swallowed and passed into the stomach and
intestines.

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 Intracellular Site of Growth
 Once mycobacteria establish themselves in tissue, they
reside principally intracellularly in monocytes,
reticuloendothelial cells, and giant cells.
 The intracellular location is one of the features that makes
chemotherapy difficult and favors microbial persistence.

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 Course of Infection and Disease
 Only 5% infected people develop clinical disease
 Untreated, the disease progresses slowly; majority
of TB cases contained in lungs
 Clinical tuberculosis divided into:
 Primary tuberculosis
 Secondary tuberculosis (reactivation or
reinfection)
 Disseminated tuberculosis

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 Primary TB
 Infectious dose 10 cells
 Phagocytosed by alveolar macrophages and multiply
intracellularly
 After 3-4 weeks immune system attacks, forming
tubercles, granulomas consisting of a central core
containing bacilli surrounded by WBCs – tubercle
 If center of tubercle breaks down into necrotic caseous
lesions, they gradually heal by calcification.

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330 jimmy ongori 2013 03/11/2013 21:19
 Secondary TB
 If patient doesn’t recover from primary tuberculosis,
reactivation of bacilli can occur.
 Tubercles expand and drain into the bronchial tubes and
upper respiratory tract.
 Gradually the patient experiences more severe symptoms.
 violent coughing, greenish or bloody sputum, fever,
anorexia, weight loss, fatigue
 Untreated, 60% mortality rate

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 Extrapulmonary TB
 During secondary TB, bacilli disseminate to regional
lymph nodes, kidneys, long bones, genital tract, brain,
and meninges.
 These complications are grave.

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 TUBERCULIN TEST
 Old tuberculin is a concentrated filtrate of broth in which tubercle bacilli have
grown for 6 weeks. A purified protein derivative (PPD) is obtained by chemical
fractionation of old tuberculin.
 Dose of Tuberculin - The volume is usually 0.1 mL injected
 Reactions to Tuberculin
 In an individual who has not had contact with mycobacteria, there is no reaction to
PPD.
 An individual who has had a primary infection develops induration, edema,
erythema in 24–48 hours, and. The skin test should be read in 48 or 72 hours.
 Considered positive if the injection of is followed by induration 10 mm or more in
diameter. Positive tests tend to persist for several days.
 The tuberculin test becomes positive within 4–6 weeks after infection (or injection
of avirulent bacilli). It may be negative in the presence of tuberculous infection
 Interpretation of Tuberculin Test
 A positive tuberculin test indicates that an individual has been infected in the
past. Itjimmy
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 Diagnostic Laboratory Tests
 A positive tuberculin test does not prove the presence of
active disease
 Specimens - fresh sputum, gastric washings, urine, pleural
fluid, cerebrospinal fluid, joint fluid, biopsy material,
blood, or other suspected material.
 Smears - Sputum, exudates, or other material is examined
for acid-fast bacilli by Ziehl-Neelsen staining. Stains of
gastric washings and urine generally are not
recommended, because saprophytic mycobacteria may be
present and yield a positive stain.
 Fluorescence microscopy is more sensitive than acid-fast
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 The sputum sample for culture is treated with NaOH
which kills other contaminating bacteria but not MTB
because cells are resistant to alkaline compounds by
virtue of their lipid layer.
 Two media are used to grow MTB - Middlebrook's
medium and Lowenstein-Jensen medium
 Culturing can take 4-6 weeks to yield visible colonies. As
a result, another method is commonly used call the
BACTEC System. Using the BACTEC system, MTB
growth can be detected in 9-16 days vs 4-6 weeks using
conventional media.

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 Management and Prevention of TB

 6-24 months of at least 2 drugs from a list of

11
 One pill regimen called Rifater (isoniazid,
rifampin, pyrazinamide)
 Vaccine based on attenuated bacilli Calmet-
Guerin strain of M. bovis used in other
countries

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 Mycobacterium leprae: The Leprosy
Bacillus
 Hansen’s bacillus/Hansen’s Disease
 Strict parasite – has not been grown on artificial media
or tissue culture
 Slowest growing of all species
 Multiplies within host cells in large packets called globi
 Causes leprosy, a chronic disease that begins in the skin
and mucous membranes and progresses into nerves

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 Epidemiology and Transmission of
Leprosy
 Endemic regions throughout the world
 Spread through direct inoculation from leprotics
 Not highly virulent; appears that health and living conditions
influence susceptibility and the course of the disease
 May be associated with specific genetic marker

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 Course of Infection and Disease
 Macrophages phagocytize the bacilli, but a weakened
macrophage or slow T cell response may not kill bacillus.
 Incubation from 2-5 years; if untreated, bacilli grow
slowly in the skin macrophages and Schwann cells of
peripheral nerves
 2 forms possible:
 tuberculoid – superficial infection without skin disfigurement
which damages nerves and causes loss of pain perception
 lepromatous – a deeply nodular infection that causes severe
disfigurement of the face and extremities

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 Diagnosing
 Combination of symptomology, microscopic examination of
lesions, and patient history
 Numbness in hands and feet, loss of heat and cold sensitivity,
muscle weakness, thickened earlobes, chronic stuffy nose
 Detection of acid-fast bacilli in skin lesions, nasal discharges, and
tissue samples

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 Treatment and Prevention
 Treatment by long-term combined therapy
 Prevention requires constant surveillance of high risk
populations.
 WHO sponsoring a trial vaccine

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 Infections by Non-Tuberculosis
Mycobacteria (NTM)
 M. avium complex – third most common cause of death in
AIDS patients
 M. kansaii – pulmonary infections in adult white males with
emphysema or bronchitis
 M. marinum – water inhabitant; lesions develop after scraping
on swimming pool concrete
 M. scrofulaceum – infects cervical lymph nodes
 M. paratuberculosis – raw cow’s milk; recovered from 65% of
individuals diagnosed with Crohn’s disease

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 Actinomycetes: Filamentous Bacilli

 Genera Actinomyces & Nocardia are nonmotile filamentous

bacteria related to mycobacteria.
 May cause chronic infection of skin and soft tissues
 Actinomyces israelii – responsible for diseases of the oral
cavity, thoracic or intestines - actinomycoses
 Nocardia brasiliensis causes pulmonary disease similar to
TB.

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 EXOTOXIN ENDOTOXIN
1. Released from the cell before 1. Integral part of cell wall
or after lysis
2. Protein
2. Endotoxin is LPS; Lipid A is toxic
3. Heat labile component
4. Antigenic and immunogenic 3. Heat stable
5. Toxoids can be produced 4. Antigenic; ??immunogenicity
6. Specific in effect on host
5. Toxoids cannot be produced
7. Produced by gram-positive and
gram-negative organisms 6. Many effects on host
7. Produced by gram-negative
organisms only

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 ENTEROBACTERIACEAE

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 IMViC Test
 Indole, Methyl Red, Voges-Prosakaur, Citrate
(IMViC) Tests:
 The tests comprise a series of important
determinations that are collectively called the
IMViC series of reactions
 The IMViC series of reactions allows for the
differentiation of the various members of
Enterobacteriaceae.

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MEDICALLY IMPORTANT ENTEROBACTERIACEAE

 Citrobacter species  Proteus spp.

 Enterobacter spp.  Salmonella spp.

 Escherichia spp.  Serratia spp.

 Klebsiella spp.  Shigella spp.

 Morganella spp.  Yersinia spp.

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 Microbiological Properties
 Gram-negative and rod shaped (bacilli)
 Ferment glucose with acid and (often) gas production
 Reduce nitrate to nitrite
 Grow on 5% sheep blood or chocolate agar in carbon
dioxide or ambient air
 Grow anaerobically (facultative anaerobes)
 Catalase positive and cytochrome oxidase negative
 Grow readily on MacConkey (MAC) and eosin
methylene blue (EMB) agars
 Grow readily at 35oC except Yersinia (25o-30oC)
 Motile by peritrichous flagella except Shigella and
Klebsiella which are non-motile
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  Somatic O antigens – these are the heat stable
polysaccharide part of the LPS.
 Flagellar H antigens – are heat labile
 Envelope or capsule K antigens – overlay the surface O
antigen and may block agglutination by O specific
antisera.
 Boiling for 15 minutes will destroy the K antigen and
unmask O antigens.
 The K antigen is called the Vi (virulence) antigen in
Salmonella typhi.

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Antigenic Structure of Enterobacteriaceae

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Escherichia coli
 Normal inhabitant of the G.I. tract.
 Some strains cause various forms of
gastroenteritis.
 Is a major cause of urinary tract infection and
neonatal meningitis and septicemia.
 May have a capsule.
 Most are motile.
 Antigenic structure - has O, H, and K antigens.
K1 has a strong association with virulence,
particularly meningitis in neonates.

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 Virulence factors
 Toxins
 Enterotoxins – produced by enterotoxigenic strains of E. coli
(ETEC). Causes a movement of water and ions from the tissues
to the bowel resulting in watery diarrhea.
 Shiga-type toxin – also called the verotoxin -produced by
enterohemorrhagic strains of E. coli (EHEC) – is cytotoxic,
enterotoxic, neurotoxic, and may cause diarrhea and ulceration
of the G.I. tract.
 Enteroaggregative toxin - (EAEC) – causes watery diarrhea.
 Hemolysins – two different types may be found: cell bound and
secreted.
 They lyse RBCs and leukocytes and may help to inhibit
phagocytosis when cell bound.
 Endotoxin
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 Adhesions – are also called colonization factors and
include both pili or fimbriae and non-fimbrial factors
involved in attachment.
 Antibodies to these may protect one from colonization.
 Virulence factors that protect the bacteria from host
defenses
o Capsule
o Outer membrane proteins - are involved in helping the
organism to invade by helping in attachment (acting as
adhesion) and in initiating endocytosis.

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 CLINICAL SIGNIFICANCE
 E coli is the most common cause of urinary tract infection and
accounts for approximately 90% of first urinary tract infections in
young women.
 Can lead to acute cystitis (bladder infection) and pyelonephritis
(kidney infection).
 S&S - urinary frequency, dysuria, hematuria, and pyuria.
 Urinary tract infection can result in bacteremia with clinical signs of
sepsis
 New evidence in women who suffer from recurrent UTIs suggests
that this is due to the formation of pod-like E. coli biofilms inside
bladder epithelial cells.
 Bacteria living on the edges of the biofilms nay break off
leading
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 Ascending urinary tract infection

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 E. coli infections
Neonatal meningitis – is the leading cause of neonatal
meningitis and septicemia with a high mortality rate.
 Usually caused by strains with the K capsular antigen.
Gastroenteritis – there are several distinct types of E. coli
that are involved in different types of gastroenteritis:
 Enterotoxigenic E. Coli (ETEC),
 Enteroinvasive E. Coli (EIEC),
 Enteropathogenic E. Coli (EPEC) ,
 enteroaggregative E. Coli (EAEC), and
 Enterohemorrhagic E. Coli (EHEC).
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 E. coli gastroenteritis
 ETEC – is a common cause of traveler’s diarrhea and
diarrhea in children in developing countries.
 The organism attaches to the intestinal mucosa via
colonization factors and then liberates enterotoxin.
 The disease is characterized by a watery diarrhea, nausea,
abdominal cramps and low-grade fever for 1-5 days.
 Transmission is via contaminated food or water.
 EPEC – Diarrhea with large amounts of mucous without
blood or pus occurs along with vomiting, malaise and low
grade fever.
 This is a problem mainly in hospitalized infants and in day
care centers.
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 E. coli gastroenteritis
 EIEC – The organism attaches to the intestinal mucosa via
pili and outer membrane proteins are involved in direct
penetration, invasion of the intestinal cells, and destruction
of the intestinal mucosa.
 Symptoms include fever, severe abdominal cramps,
malaise, and watery diarrhea followed by scanty stools
containing blood, mucous, and pus.
 EAEC – Mucous associated autoagglutinins cause
aggregation of the bacteria at the cell surface and result in
the formation of a mucous biofilm.
 Symptoms include watery diarrhea, vomiting,
dehydration and occasional abdominal pain.

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 E. coli gastroenteritis
 EHEC – The organism attaches via pili to the intestinal mucosa and
liberates the shiga-like toxin.
 Watery diarrhea progressing to bloody diarrhea and crampy
abdominal pain with no fever or low-grade fever.
Antimicrobic therapy
 E. coli is usually susceptible to a variety of chemotherapeutic
agents, The sulfonamides, ampicillin, cephalosporins,
fluoroquinolones, and aminoglycosides, though drug resistant
strains are increasingly prevalent.
 It is essential to do susceptibility testing.
 Treatment of patients with EHEC infections is not recommended
because it can increase the release of shiga-like toxins and actually
trigger HUS
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PREVENTION
 HANDWASHING after using the toilet, changing diapers
and before preparing or eating food, after contact with
animals or their environments
 COOK meats thoroughly.
 AVOID raw milk, unpasteurized dairy products, and
unpasteurized juices
 AVOID swallowing water when swimming or playing in
lakes, ponds, streams, swimming pools
 PREVENT cross contamination in food preparation areas
by thoroughly washing hands, counters, cutting boards,
and utensils after they touch raw meat.
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 Shigella species
 The natural habitat of shigellae is limited to the
intestinal tracts of humans and other primates,
where they produce bacillary dysentery.
 Contains four species that differ antigenically and, to
a lesser extent, biochemically.
 S. dysenteriae (Group A)
 S. flexneri (Group B)
 S. boydii (Group C)
 S. sonnei (Group D)
 All ferment mannitol except S. dysenteriae
 S. sonnei may show delayed lactose fermentation
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 Antigenic structure
 Differentiation into groups (A, B, C, and D) is based on O
antigen.
 Virulence factors
 Shiga toxin – is produced by S. dysenteriae and in smaller
amounts by S. flexneri and S. sonnei. This plays a role in the
ulceration of the intestinal mucosa.

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 Shigellosis - Epidemiology
 Major public health problem in many developing countries
 causes about 5 to I0% of childhood diarrhoea
 up to 25% of all diarrhea-related deaths can be associated
with Shigella
Developing countries:
 Sh. flexneri is endemic
 Sh. dysenteriae type 1 often occurs in an epidemic pattern
 These two species of Shigella generally produce the most
severe illness.
Developed countries:
 Sh. sonnei is the most common and is the least virulent
 Sh. boydii causes disease of intermediate severity
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 is least common, except in the Indian sub-continent.
 Shigellosis - Epidemiology
 Worldwide distribution; infections occur throughout year
 Mostly in children aged under five
 Rates of infection are highest where sanitation is poor
 Transmission influenced by:
 nutritional status
 environmental factors affecting transmission:
 Rainfall and temperature
 Waterwashed as well as waterborne
 Incidence highest in dryer climates in hot and dry weather
 Incidence in wet climates is often highest in rainy season

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Clinical significance
 Causes shigellosis or bacillary dysentery.
 Transmission is via the fecal-oral route.
 The infective dose required to cause infection is very low
(10-200 organisms).
 Incubation of 1-7 days followed by fever, cramping,
abdominal pain, and watery diarrhea (due to the
 This may be followed by frequent, scant stools with blood,
mucous, and pus (due to invasion of intestinal mucosa).
 It is rare for the organism to disseminate.
 The severity of the disease depends upon the species one is
infected with.
 S. dysenteria is the most pathogenic followed by S. flexneri,
S. sonnei and S. boydii.
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Treatment:
 Continue to eat (feed)
 to prevent weight loss and hypoglycemia
 include foods rich in potassium (bananas)
 Replace fluids - Oral or IV rehydration with fluids and
electrolytes
 Treat with antibiotics:
 trimethoprim/ sulfamethoxazole, Ciprofloxacin, ampicillin,
doxycycline
Prevention and Control:
 Handwashing, especially after defacation
 Improved sanitation and hygiene
 No effective vaccine
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 Salmonella
 Classification has been changing in the last few years.
 There is now 1 species: S. enteritica, and 7 subspecies: 1, 2 ,3a ,3b
,4 ,5, and 6.
 Clinically Salmonella isolates are often still reported out as
serogroups or serotypes
 Four serotypes of salmonellae that cause enteric fever can be
identified in the clinical laboratory by biochemical and serologic
tests. They are;
 Salmonella Paratyphi A (serogroup A),
 Salmonella Paratyphi B (serogroup B),
 Salmonella Choleraesuis (serogroup C1), and
 Salmonella Typhi (serogroup D).

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Antigenic Structure
 O antigens - characteristic sequence of repeating
polysaccharide units in LPS.
 H antigens - flagellar antigens (protein) and may occur in
one of two phase variations.
 Vi antigen - a capsular polysaccharide

Virulence factors
 Endotoxin – may play a role in intracellular survival
 Capsule (for S. typhi and some strains of S. paratyphi)
 Adhesions – both fimbrial and non-fimbrial

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Epidemiology
 Person to-person spread
 No animal reservoir
 Contamination with human faeces
 Usuallycontaminated water.
 Occasionally, contaminated food (usually handled
by an individual who harbours S. typhi)

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 Carrier states
 Carrier state may last from many weeks to years with
faecal shedding
 Convalescent carrier
 Chronic carrier
 ~3% of persons infected with S. typhi
 Potential for cross-contamination of foods by the infected
handler - ―Typhoid Mary‖ Mallone

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 Clinical Significance
 Causes two different kinds of disease: enteric fevers and
gastroenteritis.
 Both types of disease begin in the same way, but with the
G/E the bacteria remains restricted to the intestine and
with the enteric fevers, the organism spreads
 Transmission is via a fecal-oral route
 The organism moves through the intestinal mucosa and
adheres to intestinal epithelium.
 For gastroenteritis the Salmonella multiply and their
presence induces a strong inflammatory response which
causes most of the symptoms seen G/E
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 In enteric fevers (typhoid and paratyphoid) the Salmonella
disseminate before they multiply to high enough levels to
stimulate a strong inflammatory
 The bacteria move via the lymphatics and bloodstream to
the liver and spleen where phagocytosis and multiplication
occurs.
 The bacteria re-enter the bloodstream to disseminate
throughout the body to all organs causing fever, headaches,
myalgia, and GI problems.
 Rose spots (erythematous, muculopapular lesions) are
seen on the abdomen. Osteomyelitis, cystitis, and gall
bladder infections may occur.

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 Diagnosis of typhoid fever
 Blood cultures are positive during the first week and after
the second week
 Stool cultures and sometimes urine cultures are positive
after the second week
 The Widal test is a serological test for antibodies against
Salmonella typhi. One looks for a 4-fold rise in titer
between acute and convalescent stages.
 10% of those infected become short term carriers and a
smaller % become long-term carriers due to persistence of
the bacteria in the gallbladder or urinary bladder.
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 Differential Medium Cultures - EMB, MacConkey's, or deoxycholate
medium permits rapid detection of lactose nonfermenters Many
salmonellae produce H2S.
 Selective Medium Cultures - The specimen is plated on salmonella-
shigella (SS) agar, Hektoen enteric agar, XLD, or deoxycholate-citrate
agar, which favor growth of salmonellae and shigellae over other
Enterobacteriaceae.
 Enrichment Cultures - The specimen (usually stool) also is put into
selenite F or tetrathionate broth, both of which inhibit replication of
normal intestinal bacteria and permit multiplication of salmonellae.
After incubation for 1–2 days, this is plated on differential and selective
media.
 Serologic Methods
 Agglutination Test - known sera and unknown culture are mixed on a
slide. Clumping, when it occurs, can be observed within a few minutes.
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 Tube Dilution Agglutination Test (Widal Test) - Serum agglutinins rise
sharply during the second and third weeks of Salmonella Typhi infection.
The Widal test to detect these antibodies against the O and H antigens
has been in use for decades. At least two serum specimens, obtained at
intervals of 7–10 days, are needed to prove a rise in antibody titer.
 Serial dilutions of unknown sera are tested against antigens from
representative salmonellae.
 The interpretive criteria when single serum specimens are tested vary,
but a titer against the O antigen of > 1:320 and against the H antigen of
> 1:640 is considered positive.
 High titer of antibody to the Vi antigen occurs in some carriers. Results
of serologic tests for salmonella infection must be interpreted cautiously
because the possible presence of cross-reactive antibodies limits the use
of serology. The test is not useful in diagnosis of enteric fevers caused by
salmonella other than Salmonella Typhi.
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TREATMENT
 Gastroenteritis: Usually a self-limiting disease. Fluid

and electrolyte replacement.

 Recommended oral regimens for the treatment of

typhoid fever :
 Fluoroquinolone

 Alternatively, use of an intravenous antibiotic, such as

ceftriaxone, is effective in patients who cannot

tolerate oral medications.

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 Prevention
1. Remove source
 Salmonella free life-stock
 Vaccinate chicks
2. Interrupt transmission
Good food hygiene
 Cook food properly
 Keep raw and cooked foods apart
Public Health: clean water
3. Strengthen host
 Vaccination

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 Yersinia species
 Are short, pleomorphic gram-negative rods that can
exhibit bipolar staining.
 Catalase-positive, oxidase-negative, and microaerophilic or
facultatively anaerobic.
 Most have animals as their natural hosts, but they can
produce serious disease in humans.
 Includes;
 Yersinia pestis, the cause of plague;
 Yersinia pseudotuberculosis and Yersinia enterocolitica,
important causes of human diarrheal diseases; and others.
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 Identification
 Y. pestis can be separated from Y. enterocolitica and Y.
pseudotuberculosis by the fact that it is non-motile.
 Y. enterocolitica and Y. pseudotuberculosis are both non-motile
at 370 C, and motile at 220 C.
 Y. pestis is identified based on the following:
Non-motile
Bipolar staining
Slow growth of small colonies on ordinary culture media
– it grows better at lower temperature (25-300 C)

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 Virulence characteristics
 Endotoxin – is responsible for many of the symptoms
 Murine toxin – causes edema and necrosis in mice and rats, but
has not been shown to play a role in human disease
 V antigen – a secreted protein that controls expression of many
of the virulence factors
Pathogenesis & Pathology
 When a flea feeds on a rodent infected with Y pestis, the
ingested organisms multiply in the gut of the flea and, helped
by the coagulase, block its proventriculus so that no food can
pass through. Subsequently, the "blocked" and hungry flea bites
ferociously and the aspirated blood, contaminated with Y pestis
from the flea, is regurgitated into the bite wound.
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 Pathogenesis & Pathology
 When a flea feeds on a rodent infected with Y pestis,
ingested organisms multiply in the gut of the flea and,
helped by coagulase, block its proventriculus so that no
food can pass through. Subsequently, the "blocked" and
hungry flea bites ferociously and the aspirated blood,
contaminated with Y pestis from the flea, is regurgitated
into the bite wound.
 Inoculated organisms phagocytosed by PMN cells and
monocytes. The Y pestis organisms are killed by the
polymorphonuclear cells but multiply in the monocytes

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 Pathogenesis & Pathology
 Pathogens rapidly reach the lymphatics and an intense
hemorrhagic inflammation develops in the enlarged lymph
nodes, which may undergo necrosis.
 Reach the bloodstream and become widely disseminated.
 Hemorrhagic and necrotic lesions may develop in all
organs; meningitis, pneumonia, are prominent features.
 Primary pneumonic plague results from inhalation of
infective droplets (usually from a coughing patient), with
hemorrhagic consolidation, sepsis, and death.

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 Clinical significance
 In man plague occurs in two forms; bubonic and pneumonic
Bubonic plague – transmitted by fleas from an infected rodent
 The bacteria travel in the blood to the nearest lymph node where
they are engulfed by fixed macrophages.
 A high fever develops and the lymph nodes in the groin and
armpit become enlarged (buboes) as the bacteria proliferate and
stimulate an inflammatory response.
 The bacteria growing in the lymph node leak into the
bloodstream.
 Lysis of the bacteria releases LPS, causing septic shock.
 Subcutaneous hemorrhages, gave the disease the name, the black
death, in the middle ages.

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 Buboes

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 Eventually bacteria reach the lungs where they are
ingested by lung macrophages to cause pneumonic
plague.
Pneumonic plague – this can be transmitted directly to
others via aerosol.
 Direct inhalation of aerosols containing the organism
produces a form of the disease that progresses much
more rapidly and the mortality rate is close to 100%.

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Diagnostic Laboratory Tests
 Specimens – Blood for culture and aspirates of enlarged lymph
nodes for smear and culture, in possible meningitis CSF is taken
 Smears - Giemsa's stain and specific immunofluorescent stains.
With Wayson's stain, Y pestis shows a striking bipolar appearance.
 Culture - All materials are cultured on blood agar and
MacConkey's agar plates and in infusion broth.
 Definite identification of cultures is done by immunofluorescence
 Cultures are highly infectious and must be handled with extreme
caution.
 Serology - serum antibody titer of 1:16 or greater is presumptive
evidence of Y pestis infection. A titer rise in two sequential
specimens confirms the serologic diagnosis.
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Treatment
 Unless promptly treated, plague may have a mortality rate of
nearly 50%; pneumonic plague, nearly 100%. The drug of
choice is streptomycin. Tetracycline is an alternative drug and
is sometimes given in combination with streptomycin. Drug
resistance has been noted in Y pestis.
Epidemiology & Control
 Plague is an infection of wild rodents that occurs in many parts
of the world. Epizootics with high mortality rates occur
intermittently; at such times, the infection can spread to
domestic rodents (eg, rats) and other animals (eg, cats), and
humans can be infected by flea bites or by contact.
 The commonest vector of plague is the rat flea (Xenopsylla
cheopis), but other fleas may also transmit the infection.

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 Control;
 By destruction of plague-infected animals.
 All patients with suspected plague should be isolated
 All specimens must be treated with extreme caution.
 Contacts of patients with suspected plague pneumonia
should receive tetracycline, as chemoprophylaxis.
 A formalin-killed vaccine is available for travelers to
hyperendemic areas and for persons at special high risk.

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KLEBSIELLA-ENTEROBACTER SERRATIA GROUP
Important Properties
 Frequently found in the large intestine but are also
present in soil and water
 Group has very similar properties and are usually
disintiguished on the basis of biochemical reactions and
motility
 Klebsiella pneumoniae has a very large capsule (giving
the colonies striking mucoid appearance)
 Serratia marcescens produces red pigmented colonies

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Pathogenesis and Epidemiology
A. Klebsiella pneumoniae
 Most likely to be a primary non-opportunistic pathogen
(related to its antiphagocytic capsule)
 Predisposing factors;
 advanced age
 chronic respiratory disease
 diabetes
 alcoholism
 10% of healthy people are carriers

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B. Enterobacter /Serratia
 Associated with hospital acquired infections sec to invasive
procedures, intravenous catheterization, respiratory intubation,
urinary tract manipulation,
 Outbreaks of Serratia pnuemonia have been associated with
contamination of the water in respiratory therapy devices
 Clinical Findings/Disease
 UTI
 Bacteremiaà sepsis
 Meningitis
 Pneumonia - if due to Klebsiella, patient produces thick, bloody
sputum and can progress to necrosis and abscess formation
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Laboratory Diagnosis
 Lactose fermenting colonies on differential agar
(MacConkey’s or EMB)
Treatment
 Frequently resistant to multiple antibiotics especially if
hospital acquired
 Empiric treatment with cephalosporin plus an
aminoglycosides pending result of sensitivity testing
Prevention
 Changing site of intravenous catheters
 Removing urinary catheters when they are no longer
required
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 Genus : Proteus
 The Genus is characterized by:
 rapid urease activity (within 4 hours).
 typical swarming growth on nutrient and blood agar
plates.

 The organisms are found in soil, water and fecally

contaminated materials.

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 Clinically important species include:
1. Proteus mirabilis
 Isolated from wound and urinary tract infections.
 Indole negative.
 Susceptible to ampicillin and cephalosporins.
2. Proteus vulgaris
 Causes urinary tract infection (often nosocomial, affect
immunosuppressed patients and those receiving prolonged
antibiotic therapy).
 Indole positive.
 Resistance to both ampicillin and cephalosporins.

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Laboratory diagnosis
Specimen: urine, pus, ……etc
Microscopy: Gram-negative rods.
Culture: on blood agar medium - swarming growth.
 on MacConkey’s agar  non-lactose
fermenting colonies.
 Identification: sugar fermentation – rapid urease activity.
Treatment
According to culture & antibiotic sensitivity test results.

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Citrobacter
 Commonly associated with hospital – acquired infections
 Are opportunistic pathogens causing urinary tract or
respiratory tract infections and occasionally wound
infections, osteomyelitis, endocarditis, and meningitis.
 Especially in immunocompromised patients.
Treatment
 According to culture and antibiotic sensitivity tests results.

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 Enterobacter
 Clinical significance
 Nosocomial infections
 Bacteremia in burn patients

Serratia
 A free-living saprophyte
 Motility
 Has been found in RT and UT infections
 Is resistant to many antimicrobials

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397
 VIBRIO, CAMPYLOBACTER, AND
HELICOBACTER

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 Are gram-negative rods that are all widely distributed in
nature.
 The vibrios are found in marine and surface waters.
 The campylobacters are found in many species of animals,
including many domesticated animals. Campylobacter jejuni
is a common cause of enteritis in humans.
 Vibrio cholerae produces an enterotoxin that causes cholera,
a profuse watery diarrhea that can rapidly lead to
dehydration and death.
 Helicobacter pylori has been associated with gastritis and
duodenal ulcer disease.
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 GENUS VIBRIO
 Consists of Gram-negative straight or curved rods,
motile by means of a single polar flagellum.
 Most species are oxidase-positive.
 In most ways vibrios are related to enteric bacteria
 Vibrios are distinguished from enterics by being
oxidase-positive and motile by means of polar
flagella.
 Of the vibrios that are clinically significant to
humans, Vibrio cholerae,the agent of cholera, is the
most important.

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 Most vibrios have simple growth factor requirements and
will grow in synthetic media with glucose as a sole source
of carbon and energy. However, since vibrios are typically
marine organisms, most species require 2-3% NaCl or a
sea water base for optimal growth.
 Vibrios are one of the most common organisms in surface
waters of the world. They occur in both marine and
freshwater habitats and in associations with aquatic
animals. Some species are bioluminescent and live in
mutualistic associations with fish and other marine life.
 Other species are pathogenic for fish, eels, and frogs, as
well as other vertebrates and invertebrates.
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Antigenic Structure
 Many vibrios share a single heat-labile flagellar H
antigen.
 V cholerae has O lipopolysaccharides that confer
serologic specificity.
 The serotypes are Ogawa, Inaba, and Hikojima. Two
biotypes of epidemic V cholerae have been defined,
classic and El Tor.
Vibrio cholerae Enterotoxin
 V cholerae produce a heat-labile enterotoxin which
leads to prolonged hypersecretion of water and
electrolytes.
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 Epidemiology
 Six pandemics (worldwide epidemics) of cholera occurred between
1817 and 1923, largely originating in Asia, usually the Indian
subcontinent.
 The seventh pandemic began in 1961 in the Indonesia, with spread
to Asia, the Middle East, and Africa.
 Cholera is endemic in India and Southeast Asia. From these centers,
it is carried along shipping lanes, trade routes, and pilgrim
migration routes.
 The disease is spread by contact involving individuals with mild or
early illness and by water, food, and flies.
 Vibrios survive in water for up to 3 weeks. Vibrio cholerae lives in
aquatic environments. And such environments are the vibrios
natural reservoir.
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 Pathogenesis
 A person with normal gastric acidity may have to ingest as
many as 1010 or more V cholerae to become infected when
the vehicle is water, because the organisms are susceptible
to acid.
 When the vehicle is food, as few as 102–104 organisms are
necessary because of the buffering capacity of food. Any
medication or condition that decreases stomach acidity
makes a person more susceptible to infection with V
cholerae.
 Cholera is not an invasive infection. The organisms do not
reach the bloodstream but remain within the intestinal
tract
404
where
jimmy they multiply and liberate cholera toxin
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 Cholera
 Cholera is a severe diarrheal disease caused by the bacterium
Vibrio cholerae.
 Transmission is by water or food.
 V. cholerae produces cholera toxin, whose action on the mucosal
epithelium is responsible for the characteristic diarrhea of the
disease cholera.
 Cholera is one of the most rapidly fatal illnesses known. A healthy
person may become hypotensive within an hour of the onset of
symptoms and may die within 2-3 hours if no treatment is
provided.
 More commonly, the disease progresses from the first liquid stool
to shock in 4-12 hours, with death following in 18 hours to several
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Clinical Findings
 About 60% of infections with classic V cholerae are asymptomatic, as
are about 75% of infections with the El Tor biotype.
 Incubation period is 1–4 days
 Sudden onset of nausea and vomiting and profuse diarrhea with
abdominal cramps. Stools, which resemble "rice water," contain
mucus, epithelial cells, and large numbers of vibrios.
 Rapid loss of fluid and electrolytes, which leads to profound
dehydration, circulatory collapse, acidosis and anuria.
 Loss of fluid due to cholera enterotoxin activating the adenylate
cyclase enzyme in the intestinal cells, converting them into pumps
which extract water and electrolytes from blood and tissues and
pump it into the lumen of the intestine.
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406 and 50%.
21:19
 Diagnostic Laboratory Tests
 Specimens - mucus flecks from stools.
 Smears - The microscopic appearance of smears made from stool
samples is not distinctive. Dark-field or phase contrast microscopy
may show the rapidly motile vibrios.
 Culture
 Growth is rapid in peptone agar, on blood agar with a pH near 9.0,
or on TCBS (thiosulfate-citrate-bile-sucrose) agar, and typical
colonies can be picked in 18 hours. For enrichment, a few drops of
stool can be incubated for 6–8 hours in taurocholate-peptone broth
(pH 8.0–9.0)
 Specific Tests - slide agglutination tests using anti-O group 1 or
group 139 antisera and by biochemical reaction patterns.
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Treatment
 The most important part of therapy consists of water
and electrolyte replacement to correct the severe
dehydration and salt depletion.
 Most antibiotics and chemotherapeutic agents have no
value in cholera therapy.
 However oral tetracycline tends to reduce stool output
in cholera and shortens the period of excretion of
vibrios.

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Control
 Control rests on education and on improvement of
sanitation, particularly of food and water.
 Patients should be isolated, their excreta
disinfected, and contacts followed up.
 Chemoprophylaxis with antimicrobial drugs may
have a place.
 Repeated injection of a vaccine containing either
lipopolysaccharides extracted from vibrios or dense
vibrio suspensions can confer limited protection to
heavily exposed persons (eg, family contacts) but is
not effective as an epidemic control measure.
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Campylobacter jejuni &
Campylobacter coli
 Campylobacter jejuni and Campylobacter coli have
emerged as common human pathogens, causing
mainly enteritis and occasionally systemic infection.
 Cause infections that are clinically indistinguishable
 These bacteria are at least as common as
salmonellae and shigellae as a cause of diarrhea
 Are gram-negative rods with comma, S,
 Motile, with a single polar flagellum, and do not
form spores.
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410
Toxins
 Have lipopolysaccharides with endotoxic activity.
 Cytopathic extracellular toxins and enterotoxins have
been found
Pathogenesis & Pathology
 The infection is via oral route from food, drink, or
contact with infected animals or animal products.
 The organisms multiply in the small intestine, invade the
epithelium, and produce inflammation that results in the
appearance of red and white blood cells in the stools.
 Occasionally, the bloodstream is invaded and a clinical
picture of enteric fever develops.
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Clinical Findings
 Acute onset of crampy abdominal pain, profuse
diarrhea that may be grossly bloody, headache,
malaise, and fever.
 Usually the illness is self-limited to a period of 5–8
days, but occasionally it continues longer.
 C jejuni isolates are usually susceptible to
erythromycin, and therapy shortens the duration of
fecal shedding of bacteria. Most cases resolve
without antimicrobial therapy.

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Diagnostic Laboratory Tests
 Specimens - Diarrheal stool is the usual specimen.
 Smears - Gram-stained smears of stool may show
the typical shaped rods. Dark-field or phase
contrast microscopy may show the typical darting
motility of the organisms.
 Culture - Culture on the selective media with O2
(5% O2) with added CO2 (10% ) is the definitive test
to diagnose C jejuni enteritis

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Helicobacter pylori
 It is a spiral-shaped gram-negative rod.
 H pylori is associated with antral gastritis, duodenal (peptic)
ulcer disease, gastric ulcers, and gastric carcinoma.
 It has multiple flagella at one pole and is actively motile.
Pathogenesis & Pathology
 Grows optimally at a pH of 6.0–7.0 and would be killed or not
grow at the pH within the gastric lumen. Gastric mucus is
relatively impermeable to acid and has a strong buffering
capacity. On the lumen side of the mucus, the pH is low (1.0–
2.0) while on the epithelial side the pH is about 7.4. H pylori is
found deep in the mucous layer near the epithelial surface
where
414
physiologic
jimmy ongori 2013 pH is present 03/11/2013 21:19
Pathogenesis & Pathology Cont’
 Produces a protease that modifies the gastric mucus and
further reduces the ability of acid to diffuse through the
mucus.
 Produces urease activity, which yields ammonia and
further buffering of acid.
 There is a strong association between the presence of H
pylori infection and duodenal ulceration.
 Histologically, gastritis is characterized by chronic and
active inflammation.
 H pylori may be a major risk factor for gastric cancer.
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Clinical Findings
 Acute infection can yield an upper gastrointestinal
illness with nausea and pain; vomiting and fever
may be present also. The acute symptoms may last
for less than 1 week or as long as 2 weeks.
 Once colonized, the H pylori infection persists for
years and perhaps decades or even a lifetime.
 About 90% of patients with duodenal ulcers and
50–80% of those with gastric ulcers have H pylori
infection.
 H pylori also may have a role in gastric carcinoma
and lymphoma.
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Diagnostic Laboratory Tests
 Specimens - Gastric biopsy specimens, Blood
 Smears - The diagnosis of gastritis and H pylori
infection can be made histologically, Giemsa or
special silver stains can show the curved or
spiraled organisms.
 Antibodies - Several assays have been developed
to detect serum antibodies specific for H pylori.
 Special Tests - Rapid tests to detect urease
activity are widely used for presumptive
identification of H pylori in specimens.
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Treatment
 Triple therapy with metronidazole and either
bismuth subsalicylate or bismuth subcitrate plus
either amoxicillin or tetracycline for 14 days
eradicates H pylori infection in 70–95% of patients.
 An acid-suppressing agent given for 4–6 weeks
enhances ulcer healing. Proton pump inhibitors
directly inhibit H pylori and appear to be potent
urease inhibitors.
 Either 1 week of a proton pump inhibitor plus
amoxicillin and clarithromycin or of amoxicillin plus
metronidazole also is highly effective.
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Epidemiology & Control
 H pylori is present on the gastric mucosa of less than
20% of persons under age 30 but increases in
prevalence to 40–60% of persons age 60, including
persons who are asymptomatic.
 In developing countries, the prevalence of infection
may be 80% or higher in adults.
 Person-to-person transmission of H pylori is likely

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 SPIROCHETES

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 The spirochetes are a large, heterogeneous group of
spiral, motile bacteria.
 The family Treponemataceae includes three genera
of medical importance: Treponema, Borrelia, and
Leptospira.
 They are long, slender, helically coiled, spiral or
corkscrew-shaped, gram-negative bacilli.
 Treponemes reproduce by transverse fission.
 Axial filaments - (a form of flagella) - are the
locomotory organelles
 Cannot be cultured in vitro
jimmy ongori 2013
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Histopathology showing Treponema pallidum spirochetes in
testis of experimentally infected rabbit. Modified Steiner silver stain.
422 jimmy ongori 2013 03/11/2013 21:19
CDC/Dr. Edwin P. Ewing, Jr. [email protected]
TREPONEMA PALLIDUM
 Slender spirals measuring about 0.2 m in width
and 5–15 m in length.
 The spiral coils are regularly spaced at a distance
of 1 m from one another. The organisms are
actively motile, rotating steadily around their
endoflagella even after attaching to cells by their
tapered ends.
 The spirals are so thin that they are not readily
seen unless immunofluorescent stain or darkfield
illumination is employed.
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 Epidemiology
 Transmission: sexual contact (overwhelming
majority), kissing or other close contact with lesion,
transfusion, or direct inoculation
 Most infectious early in disease: chancre, mucous
patch, condyloma latum
 Blood transfusion rare: all donors tested and
organism cannot survive longer than 48 hrs using
current blood bank storage techniques
 Congenital infection
 12 million cases worldwide

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Culture
 Pathogenic T pallidum has never been cultured continuously on
artificial media, in fertile eggs, or in tissue culture.
 T pallidum is a microaerophilic organism; it survives best in 1–
4% oxygen.
Reactions to Physical and Chemical Agents
 Drying kills the spirochete rapidly, as does elevation of the
temperature to 42 °C.
 Killed by trivalent arsenical, mercury, and bismuth
 Penicillin is treponemicidal in minute concentrations, but the
rate of killing is slow, of slow multiplication rate of T pallidum
(estimated division time is 30 hours).
 Resistance to penicillin has not been demonstrated in syphilis
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 Virulence Factors of T. pallidum
 Outer membrane proteins promote adherence
 Hyaluronidase may facilitate perivascular
infiltration
 Antiphagocytic coating of fibronectin
 Tissue destruction and lesions are primarily
result of host’s immune response
(immunopathology)

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 Pathogenesis of T. pallidum
Tissue destruction and lesions are primarily a
consequence of patient’s immune response
Syphilis is a disease of blood vessels and of the
perivascular areas
In spite of a vigorous host immune response the
organisms are capable of persisting for decades
Infection is neither fully controlled nor eradicated
In early stages, there is an inhibition of cell-
mediated immunity
Inhibition of CMI abates in late stages of disease,
427 hence late lesions tend to be localized 03/11/2013 21:19
jimmy ongori 2013
 Primary Syphilis
 10 disease process involves invasion of mucus
membranes, rapid multiplication & wide
dissemination through perivascular lymphatics and
systemic circulation - Occurs prior to development
of the primary lesion
10-90 days after initial contact the host mounts an
inflammatory response at the site of inoculation
resulting in the hallmark syphilitic lesion, called the
chancre (usually painless)
Chancre changes from hard to ulcerative with
profuse shedding of spirochetes
428 jimmy ongori 2013 03/11/2013 21:19
 After initial infection, A primary chancre is seen in the
area of contact within 10-60 days.

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(a primary chancre is an area of ulceration/inflammation)
03/11/2013 21:19
 CHANCRE

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 syphilis

Treponema pallidum

rash gumma
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 jimmy ongori 2013
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 jimmy ongori 2013
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 Secondary Syphilis
Secondary disease 2-10 weeks after primary lesion
Widely disseminated mucocutaneous rash
Secondary lesions of the skin and mucus
membranes are highly contagious
Generalized immunological response

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 Generalized
Mucocutaneous Rash of
Secondary Syphilis

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 Latent Stage Syphilis
Following secondary disease, host enters latent
period
•First 4 years = early latent
•Subsequent period = late latent
About 40% of late latent patients progress to late
tertiary syphilitic disease

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Tertiary syphilis
 Characterized by localized granulomatous dermal lesions
(gummas) in which few organisms are present
 Granulomas reflect containment by the immunologic
reaction of the host to chronic infection
 Late neurosyphilis develops in about 1/6 untreated cases,
usually more than 5 years after initial infection
• Central nervous system and spinal cord involvement
• Dementia, seizures, wasting, etc.
 Cardiovascular involvement appears 10-40 years after
initial infection with resulting myocardial insufficiency
and death
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 Congenital Syphilis
 A pregnant syphilitic woman can transmit T
pallidum to the fetus through the placenta
beginning in the 10th to 15th weeks of gestation.
 Some of the infected fetuses die, and miscarriages
result; others are stillborn at term.
 Others are born live but develop the signs of
congenital syphilis in childhood: interstitial
keratitis, Hutchinson's teeth, saddlenose,
periostitis, and a variety of central nervous system
anomalies. Adequate treatment of the mother
during pregnancy prevents congenital syphilis.
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Diagnostic Laboratory Tests
 Specimens - tissue fluid from early surface lesions
for demonstration of spirochetes; blood serology
 Darkfield Examination - examined under oil
immersion with darkfield illumination for typical
motile spirochetes.
 Treponemes disappear from lesions within a few hours
after the beginning of antibiotic treatment.
 Immunofluorescence - for typical fluorescent
spirochetes.
 Serologic Tests for Syphilis (STS) - These tests
usejimmy
either nontreponemal or treponemal antigens
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 Treponemal Antibody Tests
 Fluorescent Treponemal Antibody (FTA-ABS) Test - It is the
first to become positive in early syphilis, is routinely
positive in secondary syphilis, and usually remains positive
many years after effective treatment. The presence of IgM
FTA in the blood of newborns is good evidence of in utero
infection (congenital syphilis).
 Treponema pallidum-Particle Agglutination (TP-PA) Test
 The most widely used nontreponemal antibody tests for
syphilis are the RPR and VDRL tests, which measure IgG
and IgM
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 Prevention & Treatment of Syphilis

 Penicillin (benzathine pen.) remains drug of

choice
• WHO monitors treatment recommendations
• 7-10 days continuously for early stage
• At least 21 days continuously beyond the
early stage
 Prevention with barrier methods (e.g., condoms)
 Prophylactic treatment of contacts identified
through epidemiological tracing
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 LEPTOSPIRAE
 Are tightly coiled, thin, flexible spirochetes 5–15 m long, with
very fine spirals 0.1–0.2 m wide; one end is often bent,
forming a hook.
 Actively motile, best seen using a darkfield microscope.
 It does not stain
Culture
 Grow best under aerobic conditions at 28–30 °C in serum-
containing semisolid media. After 1–2 weeks the leptospirae
produce a diffuse zone of growth near the top of the tube and
later a ring of growth at a level in the tube corresponding to
the level of the optimal oxygen tension for the organisms. T
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 Leptospira interrogans
 Leptospires are coiled, thin, highly motile organisms
with hooked ends and two periplasmic flagella that
permit burrowing into tissue.
 They stain poorly but can be seen microscopically by
dark-field examination
 Leptospires require special media and conditions for
growth; it may take weeks for cultures to become
positive.

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443
Epidemiology
 Leptospirosis is an important zoonosis with a worldwide
distribution
 Rodents, esp rats are the most important reservoir, other wild
mammals , domestic and farm animals may also harbor it
 Occurs most commonly in the tropics because the climate as well
as the sometimes-poor hygienic conditions favor the pathogen's
survival and distribution.
 Transmission of leptospires to humans may follow direct contact
with urine, blood, or tissue from an infected animal or exposure to
a contaminated environment; human-to-human transmission is
rare.
 Since leptospires can survive in water for many months, water is
an important
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Pathogenesis & Clinical Findings
 Infection results from leptospires entering the body through breaks
in the skin and mucus membranes (mouth, nose, conjunctivae).
 After an incubation period of 1–2 weeks, there is a variable febrile
onset during which spirochetes are present in the bloodstream.
 They establish themselves in the organs (particularly liver and
kidneys), producing hemorrhage and necrosis of tissue and
resulting in dysfunction eg jaundice.
 After initial improvement, the 2nd phase develops, It manifests
itself often as "aseptic meningitis" with intense headache, stiff
neck, and pleocytosis of the cerebrospinal fluid.
 Nephritis and hepatitis may also recur, and there may be skin,
muscle, and eye lesions.
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445
  Weil's syndrome, the most severe form of
leptospirosis, is characterized by jaundice,
renal dysfunction, and hemorrhagic diathesis;
by pulmonary involvement in many cases; and
by mortality rates of 5–15%

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Diagnostic Laboratory Tests
 Specimens - blood, cerebrospinal fluid, or tissues Urine
should be collected using great care to avoid
contamination.
 Microscopic Examination - Darkfield examination or
thick smears stained by the Giemsa technique
 Culture - Whole fresh blood or urine can be cultured in
Fletcher's semisolid or other medium.
 Animal Inoculation - intraperitoneal inoculation of
guinea pigs with fresh plasma or urine. Within a few days,
spirochetes become demonstrable in the peritoneal cavity;
on the death of the animal (8–14 days), hemorrhagic
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 Treatment
 Mild leptospirosis - oral doxycycline, ampicillin, or
amoxicillin, moderate or severe disease should be IV penicillin
Prevention, & Control
 These is animal infection; human infection is only accidental,
Rats, mice, wild rodents, dogs, swine, and cattle are the
principal sources of human infection.
 Leptospirae remain viable in stagnant water for several weeks;
drinking, swimming, bathing, or food contamination may lead
to human infection.
 Control - preventing exposure to potentially contaminated
water and rodent control. Doxycycline, 200 mg once weekly
during
jimmyheavy
ongori 2013exposure, is effective prophylaxis.
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 The Haemophilus Species
 Gram-negative, pleomorphic bacteria that require
enriched media, usually containing blood or its
derivatives, for isolation.
 Obligate Parasites of Man and Animals

 Haemophilus influenzae type b is an important

human pathogen;
 Haemophilus ducreyi, a sexually transmitted
pathogen, causes chancroid

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449
 Haemophilus influenzae
 Found on the mucous membranes of the upper respiratory
tract in humans. An important cause of meningitis in children
and occasionally causes RTI children and adults.
 The organisms are short, coccoid bacilli, sometimes occurring
in pairs or short chains.
 Have a capsule.
Culture
 On chocolate agar, flat, grayish-brown colonies are present
after 24 hours of incubation.
 H influenzae does not grow on sheep blood agar except around
colonies of staphylococci ("satellite phenomenon").
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Antigenicity
 Encapsulated H influenzae contains capsular polysaccharides of
one of six types (a–f). - The capsular antigen
 The somatic antigens of H influenzae consist of outer membrane
proteins.
Pathogenesis
 The capsule is antiphagocytic , The capsular antigen of type b H
influenzae is the major virulence factor.
 Type b H influenzae causes meningitis, pneumonia and empyema,
epiglottitis, cellulitis, septic arthritis, and occasionally other forms
of invasive infection.
 The blood of many persons over age 3–5 years is bactericidal for H
influenzae, and clinical infections are less frequent in such
individuals.
451
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Clinical Findings
 Enters through the respiratory tract. There may be
local extension with involvement of the sinuses or
the middle ear. H influenzae type b and
pneumococci are two of the most common etiologic
agents of bacterial otitis media and acute sinusitis.
 Reach the bloodstream and carried to the meninges
or, in the joints to produce septic arthritis.
 Prior to the use of the conjugate vaccine, H
influenzae was the most common cause of bacterial
meningitis in children age 5 months to 5 years in the
United States.
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  Occasionally, a fulminating obstructive
laryngotracheitis with swollen, cherry-red
epiglottis develops in infants
 Pneumonitis and epiglottitis due to H
influenzae may follow upper respiratory tract
infections in small children and old or
debilitated people. Adults may have bronchitis
or pneumonia due to H influenzae.

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Diagnostic Laboratory Tests
 Specimens - nasopharyngeal swabs, pus, blood, and
spinal fluid for smears and cultures.
 Direct Identification - Commercial kits are available
for immunologic detection of H influenzae antigens in
spinal fluid.
 Culture
 Specimens are grown on IsoVitaleX-enriched chocolate
agar until typical colonies appear. H influenzae is
differentiated from related gram-negative bacilli by its
requirements for X (heme) and V (nicotinamide-adenine
dinucleotide) factors and by its lack of hemolysis on blood
agar
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Treatment
 The mortality rate of untreated H influenzae
meningitis may be up to 90%.
 Many strains of H influenzae type b are susceptible
to ampicillin, but up to 25% produce -lactamase
under control of a transmissible plasmid and are
resistant.
 Essentially all strains are susceptible to the third-
generation cephalosporins eg IV Cefotaxime

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Epidemiology, Prevention, & Control
 Encapsulated H influenzae type b is transmitted
from person to person by the respiratory route.
 Disease can be prevented by administration of
Haemophilus b conjugate vaccine to children.
Given in Kenya as the Hib component of
Pentavalent vaccine at 6, 10 and 14 weeks of life
 Contact with patients suffering from H influenzae
clinical infection poses little risk for adults but
presents a definite risk for nonimmune siblings and
other nonimmune children under age 4 years who
are close contacts.
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 BRUCELLAE

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 Small, Gram -VE cocobacilli, non-motile, non-spore
forming.
 Brucellae grow aerobically.
 Some spp. Require supplemental carbon dioxide for
primary isolation.
 Any peptone-based media enriched with blood or serum
serve for in vitro cultivation.
 Isolation form clinical specimens require prolonged (≥ 30
days) incubation.
 Brucella strains always catalse-positive; but oxidase and
urease
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 Genus Brucella divided into six spp. on basis of
preferred hosts and cultural, metabolic and
antigenic characteristics.
Epidemiology
 Brucellosis – zoonosis – all infections, derive directly
or indirectly from animal exposure.
 Disease exists world-wide
 B. abortus found mainly in cattle, but others spp.
like buffalo, camels
 B. Melitensis primary affects goats and sheep.
Camels can be important source in some countries.

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 B. Suis in domestic and swine, cause abattoir-assoc.
human disease.
 B. Canis: Least common cause of human disease.
 Animals: Brucellosis, Chronic Infection, persisting
for life.
 Brucellae localization in reproductive organs,
accounts for major manifestations – abortion and
sterility.
 Brucellae shed in large numbers in: Milk, urine,
products of infected animals.

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 Brucellosis constitutes occupational risk for:
farmers, veterinarians, abattoirs and Laboratory
personnel.
 Routes of transmission to human include:
- Direct contact with animals or their secretions,
through cuts and skin abrasions.
- Infected aerosols inhaled or inoculated into eye
conjunctival sac.
- Ingestion of unpasteurized dairy products.

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 - Meat products: rare source of infection
because meat is rarely eaten raw and
organisms are present in low number of
muscle tissue.
- Blood and bone marrow may transmit
disease when ingested in some cultures.
- Human-to-human transmission: Unusual, but
rare cases suspected to be sexually transmitted.

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Pathogenesis
 B. melitensis and B. suis, more virulent than B.
abortus and B. canis.
 Infection with any B. species, can cause serious
human disease.
 Once brucellae gain entry to body: PMN –
Leukocytes attracted to inoculation site by
chemotaxis.
 Normal human serum has limited bactericidal
activity against brucellae, but it effectively opsonizes
bacteria for phagocytosis by PMN – Leukocytes.
 Are Facultative intracellular, slowly dividing
pathogens with capacity to survive and multiply
within host phagocytic cells.
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 Hematogenous dissemination then followed by
localization of bacteria within organs rich in
reticuloendothelial system, e.g. liver, spleen and B.
marrow.

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Clinical Manifestations
 Fever, sweats, malaise, anorexia, headache,
backpain.
 Onset: acute or insidious, beginning within 2 to 4
weeks after inoculation.
 Malodorous sweat and peculiar mouth taste.
 Depression common.
 In comparison to plethora of somatic complaints,
physical abnormalities are few.
 Mild lymphadenopathy reported in 10 to 20% of
cases.
 Splenomegaly or hepatomegaly in 20 to 30% of
cases.
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 Diagnostic Laboratory Tests
Culture - Brucella agar was specifically designed to
culture Brucella species - Bone marrow and blood
are the specimens from which brucellae are most
often isolated.
- Most Labs. Now use rapid isolation techniques, e.g.
BACTEC
 Agglutination Test - IgG agglutinin titers above 1:80
indicate active infection.
 Rose Bengal test

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 Treatment
 Brucellae may be susceptible to tetracyclines or
ampicillin. Symptomatic relief may occur within
a few days after treatment with these drugs is
begun. However, because of their intracellular
location, the organisms are not readily
eradicated completely from the host. For best
results, treatment must be prolonged. Combined
treatment with a tetracycline (such as
doxycycline) and either streptomycin for 2–3
weeks or rifampin for 6 weeks is recommended.

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Prevention
 Prevention of human brucellosis depend on:
Control and elimination of Brucellosis in domestic
animals.
 Effective attenuated live bacterial vaccines exist for:
- B. abortus
- B. melitensis
- No vaccines for B. suis and B. canis
 No licensed human vaccine

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 Bordetella Pertussis
 Gram –VE Coccobaccili (rod-shaped)
 Obligate aerobe
 Optimum growth 350C-370 C (mesophile)
 Colonizes the respiratory tract
Whooping Cough (Pertussis)
 Specific to human hosts
 Approximately the size of 0.8 µm by 0.4 µm.
 pH - Found to grow better on media with a slightly
acidic reaction

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 Virulence Factors
 Adhesions - Filamentous hemagglutinin (FHA),
Pertactin, Fimbriae
 Toxins
Pertussis Toxin (PTX) - Colonizing factor
Adenylate Cyclase Toxin (CYA) - Invasive toxin
Tracheal Cytotoxin (TCT)

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Clinical Findings
 Incubation period about 2 weeks, the "catarrhal
stage" develops, with mild coughing and sneezing.
During this stage, large numbers of organisms are
sprayed in droplets, and the patient is highly
infectious
 "paroxysmal" stage - the cough develops its
characteristic "whoop" upon inhalation. This leads
to rapid exhaustion and may be associated with
vomiting, cyanosis, and convulsions. The "whoop"
and major complications occur predominantly in
infants; paroxysmal coughing predominates in older
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Diagnostic Laboratory Tests
 Specimens - A saline nasal wash is the preferred specimen.
 Direct Fluorescent Antibody (FA) Test
 Culture - cultured on solid medium agar.
 PCR is the most sensitive method
Treatment
 Erythromycin during the catarrhal stage of disease
promotes elimination of the organisms and may have
prophylactic value.
 Treatment after onset of the paroxysmal phase rarely alters
the clinical course.
 Oxygen inhalation and sedation may prevent anoxic damage
to the brain.
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Prevention
 Every infant should receive three injections of
pertussis vaccine during the first year of life
 In Kenya Pertussis vaccine is usually administered in
combination with toxoids of diphtheria and tetanus
and Hepatitis B and Haemophilus influenza type B as
Pentavalent vaccine at 6, 10 and 14 weeks.
 Prophylactic administration of erythromycin for 5
days may also benefit unimmunized infants or
heavily exposed adults.

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 CHLAMYDIA
 Chlamydiae that infect humans are divided into
three species—Chlamydia trachomatis,
Chlamydophila (Chlamydia) pneumoniae, and
Chlamydophila (Chlamydia) psittaci
 In chlamydiae, the outer cell wall resembles the cell
wall of gram-negative bacteria. It has a relatively
high lipid content. It is rigid but does not contain a
typical bacterial peptidoglycan.
 Lysozyme has no effect on chlamydial cell walls.

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474
 Obligatory intracellular bacteria
 Infect columnar epithelial cells
 Survive by replication that results in the death of the
cell
 Takes on two forms in its life cycle:
 Elementary body (EB)
 Reticulate body (RB)

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 Pathology

Chlamydiaceae Family
(species that cause disease in humans)

Species (genus) Disease

Trachoma, NGU,
C. trachomatis MPC, PID,
2 biovars, non-LGV conjunctivitis,
LGV Infant pneumonia,
LGV
Pharyngitis,
C. pneumoniae bronchitis,
pneumonia

C. psittaci Psittacosis

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Staining Properties
 Chlamydiae have distinctive staining properties.
Elementary bodies stain purple with Giemsa stain,
the larger, noninfective reticulate bodies stain blue
with Giemsa stain.
 The Gram reaction of chlamydiae is negative or
variable and is not useful in identification of the
agents.

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Characteristics of Host-Parasite Relationship
 The outstanding biologic feature of infection by chlamydiae
is the balance that is often reached between host and
parasite, resulting in prolonged persistence of infection.
Subclinical infection is the rule—and overt disease the
exception—in the natural hosts of these agents.
 Antibodies to several antigens of chlamydiae are produced
by the infected host. These antibodies have little protective
effect against reinfection. The infectious agent commonly
persists in the presence of high antibody titers. Treatment
with effective antimicrobial drugs for prolonged periods
may eliminate the chlamydiae from the infected host.
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Clinical Syndromes Caused by C. trachomatis

Local Infection Complication Sequelae

Conjunctivitis Chronic arthritis
Men Reiter’s syndrome
Urethritis (rare)
Epididymitis
Prostatitis Infertility (rare)
Infertility
Conjunctivitis Endometritis
Ectopic pregnancy
Women Urethritis Salpingitis
Chronic pelvic pain
Cervicitis Perihepatitis
Chronic arthritis
Proctitis Reiter’s syndrome
(rare)
Conjunctivitis
Infants Chronic lung
Pneumonitis Rare, if any
Pharyngitis disease?
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 C. trachomatis Infection in Men
 Urethritis–One cause of non-gonococcal urethritis
(NGU)
 Majority (>50%) asymptomatic
 Symptoms/signs if present: mucoid or clear
urethral discharge, dysuria
 Incubation period unknown
Complicates to;
 Epididymitis
 Reiter’s Syndrome - Rarely occurs in women
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 C. trachomatis Infections in Women
 Cervicitis - Majority (70%-80%) are asymptomatic
 Local signs of infection, when present, include:
 Mucopurulent endocervical discharge
 Urethritis - Usually asymptomatic
 Complications - Pelvic Inflammatory Disease (PID)
(Salpingitis, Endometritis)
 Reiter’s Syndrome

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 C. trachomatis Infections
in Infants
 Perinatal clinical manifestations:
 Inclusion conjunctivitis
 Pneumonia

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 Diagnosis

Diagnosis
 Culture
 Non-culture tests
 Nucleic Acid Amplification Tests (NAATs)
 Non-Nucleic Acid Amplification Tests (Non-
NAATs)
 Serology

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Lymphogranuloma Venereum
 LGV is a sexually transmitted disease caused by C trachomatis and
characterized by suppurative inguinal adenitis
 Clinical Findings - Several days to several weeks after exposure,
a small, evanescent papule or vesicle develops on any part of the
external genitalia, anus, rectum, or elsewhere. The lesion may
ulcerate, but usually it remains unnoticed and heals in a few days.
Soon thereafter, the regional lymph nodes enlarge and tend to
become matted and painful.
 The nodes suppurate and discharge pus. In females and in
homosexual males, the perirectal nodes are involved, with proctitis
and a bloody mucopurulent anal discharge.
 chronic inflammatory process progresses to fibrosis, lymphatic
obstruction, and rectal strictures. The lymphatic obstruction may
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484
 VIRUSES

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 DNA VIRUSES

Parvoviridae

Hepadnaviridae

Papillomaviridae

Adenoviridae

Herpesviridae

poxviridae

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 RNA VIRUSES

PICORNAVIRIDAE

CALICIVIRIDAE

Adenoviridae

Herpesviridae

poxviridae

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Introduction
 Viruses are the smallest infectious agents and contain
only one kind of nucleic acid (RNA or DNA) as their
genome.
 The nucleic acid is encased in a protein shell, which may
be surrounded by a lipid-containing membrane. The
entire infectious unit is termed a virion.
 Viruses are inert in the extracellular environment; they
replicate only in living cells, being parasites at the
genetic level.
 The viral nucleic acid contains information necessary for
programming the infected host cell to synthesize virus-
specific macromolecules required for the production of
viral progeny.
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  During the replicative cycle, numerous copies of viral nucleic
acid and coat proteins are produced. The coat proteins assemble
together to form the capsid, which encases and stabilizes the viral
nucleic acid against the extracellular environment and facilitates
the attachment and penetration by the virus upon contact with
new susceptible cells. The virus infection may have little or no
effect on the host cell or may result in cell damage or death.
 The universe of viruses is rich in diversity. Viruses vary greatly in
structure, genome organization and expression, and strategies of
replication and transmission. The host range for a given virus may
be broad or extremely limited. Viruses are known to infect
unicellular organisms such as mycoplasmas, bacteria, and algae
and all higher plants and animals.

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Terms & Definitions in Virology
 Capsid: The protein shell, or coat, that encloses the
nucleic acid genome.
 Capsomeres: Morphologic units seen in the electron
microscope on the surface of icosahedral virus particles.
 Defective virus: A virus particle that is functionally
deficient in some aspect of replication.
 Envelope: A lipid-containing membrane that surrounds
some virus particles. It is acquired during viral
maturation by a budding process through a cellular
membrane.
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 Nucleocapsid: The protein-nucleic acid complex
representing the packaged form of the viral genome.
 Structural units: The basic protein building blocks of the
coat. They are usually a collection of more than one
nonidentical protein subunit.
 Subunit: A single folded viral polypeptide chain.
 Virion: The complete virus particle. In some instances
(eg, papillomaviruses, picornaviruses), the virion is
identical with the nucleocapsid. In more complex virions
(herpesviruses, orthomyxoviruses), this includes the
nucleocapsid plus a surrounding envelope. This structure,
the virion, serves to transfer the viral nucleic acid from
one
491 cell to another.
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 jimmy ongori 2013
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 How are viruses named?
 Based on:
- the disease they cause
poliovirus, rabies virus
- the type of disease
murine leukemia virus
- geographic locations
Sendai virus, Coxsackie virus
- their discovers
Epstein-Barr virus
- how they were originally thought to be contracted
dengue virus (―evil spirit‖), influenza virus (the ―influence‖ of bad air)
- combinations of the above
Rous Sarcoma virus

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Viral structure
 Certain viruses contain ribonucleic acid (RNA), while other viruses
have deoxyribonucleic acid (DNA). The nucleic acid portion of the
viruses is known as the genome. The nucleic acid may be single-
stranded or double-stranded; it may be linear or a closed loop; it
may be continuous or occur in segments.
 The genome of the virus is surrounded by a protein coat known as a
capsid, which is formed from a number of individual protein
molecules called capsomeres. Capsomeres are arranged in a
precise and highly repetitive pattern around the nucleic acid. A
single type of capsomere or several chemically distinct types may
make up the capsid. The combination of genome and capsid is called
the viral nucleocapsid.
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 A number of kinds of viruses contain envelopes. An envelope is a
membrane like structure that encloses the nucleocapsid and is
obtained from a host cell during the replication process. The
envelope contains viral-specified proteins that make it unique.
Enveloped viruses - herpes simplex, chickenpox, and infectious
mononucleosis etc
 The nucleocapsids of viruses are constructed according to certain
symmetrical patterns. The virus that causes tobacco mosaic disease,
for example, has helical symmetry. In this case, the nucleocapsid
is wound like a tightly coiled spiral. Other viruses take the shape of
an icosahedron, and they are said to have icosahedral symmetry.
In an icosahedron, the capsid is composed of 20 faces, each shaped
as an equilateral triangle. Among the icosahedral viruses are those
that cause yellow fever, polio, and head colds.
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 Bacteriophages;
 Are viruses that multiply within bacteria. These
viruses are among the more complex viruses. They
often have icosahedral heads and helical tails.
 Bacteriophages contain DNA and are important
tools for viral research.

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 VIRAL REPLICATION
Viral life cycle consists of six stages within the host
cell
 Attachment
 Penetration
 Uncoating
 Multiplication
 Assembly
 Release

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 During the process of viral replication, a virus induces a
living host cell to synthesize the essential components for
the synthesis of new viral particles. The particles are then
assembled into the correct structure, and the newly
formed virions escape from the cell to infect other cells.
1. The first step in the replication process is attachment. In
this step, the virus adsorbs to a susceptible host cell. High
specificity exists between virus and cell, and the envelope
spikes may unite with cell surface receptors. Receptors
may exist on bacterial pili or flagella or on the host cell
membrane.

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2. The next step is penetration of the virus or the viral genome
into the cell. This step may occur by phagocytosis; or the
envelope of the virus may blend with the cell membrane; or
the virus may ―inject‖ its genome into the host cell. The latter
situation occurs with the bacteriophage when the tail of the
phage unites with the bacterial cell wall and enzymes open a
hole in the wall. The DNA of the phage penetrates through this
hole.
3. The replication steps of the process occur next. The protein
capsid is stripped away from the genome, and the genome is
freed in the cell cytoplasm. If the genome consists of RNA, the
genome acts as a messenger RNA molecule and provides the
genetic codes for the synthesis of enzymes. The enzymes are
used for the synthesis of viral genomes and capsomeres and the
assembly of these components into new viruses.
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 If the viral genome consists of DNA, it provides the
genetic code for the synthesis of messenger RNA
molecules, and the process proceeds.
 In some cases, such as in HIV infection, the RNA of
the virus serves as a template for the synthesis of a
DNA molecule. The enzyme reverse transcriptase
catalyzes the DNA's production. The DNA molecule
then remains as part of the host cell's chromosome
for an unspecified period. From this location, it
encodes messenger RNA molecules for the synthesis
of enzymes and viral components.

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4. Once the viral genomes and capsomeres have been
synthesized, they are assembled to form new virions.
This assembly may take place in the cytoplasm or in
the nucleus of the host cell. After the assembly is
complete, the virions are ready to be released into
the environment.

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5. For the release of new viral particles, any of a
number of processes may occur. For example, the
host cell may be “biochemically exhausted,” and it
may disintegrate, thereby releasing the virions.
 For enveloped viruses, the nucleocapsids move
toward the membrane of the host cell, where they
force themselves through that membrane in a
process called budding. During budding, a portion of
cell membrane pinches off and surrounds the
nucleocapsid as an envelope. The replication
process in which the host cell experiences death is
called the lytic cycle of reproduction.
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 Lysogeny. Not all viruses multiply by the lytic cycle
of reproduction. Certain viruses remain active
within their host cells for a long period without
replicating. This cycle is called the lysogenic cycle.
 An example of lysogeny occurs in HIV infection. In
this case, the human immunodeficiency virus
remains latent within the host T-lymphocyte. An
individual whose infection is at this stage will not
experience the symptoms of AIDS until a later date.

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 Viruses infect all major groups of organisms: vertebrates, invertebrates,
plants, fungi, bacteria but some viruses have a broader host range than
others; however, none can cross the eukaryotic/prokaryotic boundary.
Factors that affect host range include:
 Whether the virus can get into the host cell; that is, does it have the
correct attachment protein to bind to a receptor on the cell surface? For
example, HIV is largely restricted to cells that have the CD4 antigen on
their surface
 Whether the appropriate cellular machinery is available for the virus to
replicate; for example, some DNA viruses can only replicate in dividing
cells which have high enough levels of deoxyribonucleotides for viral
DNA synthesis.
 If the virus can replicate, whether infectious virus can get out of the cell
and spread the infection.
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STRUCTURE
Viruses can have the following shapes;
1. Helical symmetry
2. Icosahedral symmetry
 An icosahedron is a solid with twenty faces . All
faces of the icosahedron are identical.
3. Helical symmetry
4. Complex symmetry

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FIVE BASIC STRUCTURAL FORMS OF VIRUSES IN
NATURE
1. Naked icosahedral e.g. poliovirus, adenovirus,
hepatitis A virus
2. Naked helical e.g. tobacco mosaic virus. So far no
human viruses with this structure are known
3. Enveloped icosahedral e.g. herpes virus, yellow fever
virus, rubella virus
4. Enveloped helical e.g. rabies virus, influenza virus,
parainfluenza virus, mumps virus, measles virus
5. Complex e.g. poxvirus
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 UNCONVENTIONAL AGENTS
 There are also the 'unconventional agents' sometimes
known as 'unconventional viruses' or 'atypical viruses' - Up
to now, the main kinds that have been studied are viroids
and prions.
1. VIROIDS
 Viroids contain RNA only. They are small (less than 400
nucleotides), single stranded, circular RNAs. The RNAs
are not packaged, do not appear to code for any proteins,
and so far have only been shown to be associated with plant
disease. However, there are some suggestions that
somewhat similar agents may possibly be involved in some
human
509 diseases.
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 Hepatitis delta virus
 At present, the only known human disease agent to
resemble viroids is hepatitis delta virus (HDV).
 HDV has a very small RNA genome compared to most
viruses, although it is somewhat larger than viroids.
2. PRIONS
 Prions contain protein only. They are small, proteinaceous
particles and there is controversy as to whether they
contain any nucleic acid, Examples of prion-caused human
diseases are Kuru, Creutzfeldt-Jakob disease and
Gerstmann-Straussler syndrome.
 Prions
jimmyalso cause scrapie in sheep.
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 LENTIVIRUSSES

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 Characteristics:
 Members are nononcogenic and may be cytocidal
 Infect cells of the immune system
 Proviruses remain permanently associated with cells
 Cause slowly progressive, chronic diseases
 Replication is usually species-specific
 Group includes the causative agents of AIDS

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 Disinfection & Inactivation
 HIV is completely inactivated by treatment for 10
minutes at room temperature with any of the
following: 10% household bleach, 50% ethanol, 35%
isopropanol, 1% Nonidet P40, 0.5% Lysol, 0.5%
paraformaldehyde, or 0.3% hydrogen peroxide. The
virus is also inactivated by extremes of pH (pH 1.0,
pH 13.0).
 HIV is readily inactivated in liquids or 10% serum by
heating at 56 °C for 10 minutes, but dried
proteinaceous material affords marked protection.
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HUMAN IMMUNODEFICIENCY VIRUS AND AIDS
 The world pandemic of AIDS has been with us for more than
twenty five years and shows no signs of abatement. Three million
people around the world die of AIDS each year and, so far, more
than 25 million people have died of the disease. Today, at least 33
million people are infected and there are more than 14,000 new
infections every day.
 AIDS is caused by Human Immunodeficiency Virus (HIV) which is
found in all cases of the disease. The primary targets of HIV are
activated CD4+ T4 helper lymphocytes but the virus can also infect
several other cell types including macrophages. It is the loss of T4
helper lymphocytes that leads to immunosuppression in the patient
and the consequent fatal opportunistic infections.
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514
 HIV is a lentivirus, a class of retrovirus. The name
lentivirus means slow virus, so called because these viruses
take a long time to cause overt disease.
 Most lentiviruses target cells of the immune system and
thus disease is often manifested as immunodeficiency.
 There are five known serogroups of lentivirus that infect
primates, sheep and goats, horses, cats, and cattle.

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 Two types of HIV: HIV-1 and HIV-2. These cause
clinically indistinguishable disease, although the time to
disease onset is longer for HIV-2.
 The worldwide epidemic of HIV and AIDS is caused by
HIV-1 while HIV-2 is mostly restricted to west Africa.
 Lentiviruses integrate into the host cell genome as a
provirus in the same manner as other retroviruses
 HIV can lie dormant in the proviral form within a cell for
many years, especially in resting (memory) CD4+ T4
lymphocytes, and may set up a lifelong infection. When
these cells become reactivated, viral production occurs
againjimmy
and ultimately destroys the cell.
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 Although HIV may disappear from the cells of the circulation,
replication and budding continue to occur in other tissues in the
absence of chemotherapy.
 HIV can be detected by the presence of anti-HIV antibodies or by
the presence of the virus itself using (PCR) that detects viral RNA.
 From the original infection, there is usually a period of 8 to 10
years before the clinical manifestations of AIDS occur; however, this
period may be two years or less. Approximately 10% of patients
succumb to AIDS within 2 to 3 years.
 From the original infection, there is usually a period of 8 to 10
years before the clinical manifestations of AIDS occur; however, this
period may be two years or less. Approximately 10% of patients
succumb to AIDS within 2 to 3 years.
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 Acute infection (acute retroviral syndrome)
 This period lasts for 6 to 12 weeks after initial infection until anti-
HIV antibodies are detectable.
 If acquired by sexual activity, the virus enters the body in infected
macrophages in semen or vaginal secretions. Dendritic cells in the
mucosal linings bind the virus shed by macrophages and carry it to
the lymph nodes where CD4+ T4 cells become infected. During the
course of the disease, the virus migrates to other cell types.
 Initially, HIV infection produces a mild disease that is self-
limiting. This is not seen in all patients and about 30% remain
asymptomatic during the initial period of infection. In the period
immediately after infection, virus titer rises (about 4 to 11 days
after infection) and continues at a high level over a period of a few
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 Patient experiences some symptoms (fever, rash, swollen lymph
glands) but none of these is life-threatening. There is an initial fall in
the number of CD4+ cells and a rise in CD8+ cells but they quickly
return to near normal. At this stage virus titers are very high with
as many as one hundred million virus particles per milliliter of
plasma.
 There is a "window period" of seronegativity during which an
infected person does not give a positive western blot HIV test or
ELISA, even though the viral load is high and the patient may
exhibit some symptoms.
 This seronegative period can last for six months before
seroconversion although the latter usually occurs between one and
four weeks after infection.
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A strong cell-mediated and humoral anti-HIV immune defense
 Cytotoxic B and T lymphocytes mount a strong defense and virus largely
disappears from the circulation. After the increased cell-mediated
immune response, there is a rise in humoral antibodies. During this
period of strong immune response to the virus, more than 10 billion
new HIV particles are produced each day but they are rapidly cleared by
the immune system and have a half life of only 5 to 6 hours.
 At this stage, most of this virus is coming from recently infected
proliferating CD4+ cells.Thus, the virus is destroying the very cells that
are proliferating to protect against it. The infected cells that are
producing this virus are destroyed either by the immune system or by
the virus and have a half life about 1 day. However, the rate of
production of CD4+ cells can compensate for the loss of cells and a
steady state is set up in which most CD4+ cells are uninfected.
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 Although activated, proliferating CD4+ cells are destroyed
by the immune system, a small fraction of the infected cells
survive long enough to revert back to the resting memory
state (as do non-infected CD4+ memory cells). The resting
memory cells (also referred to as anamnestic T cells) do
not express viral antigens but do carry a copy of the HIV
genome which remains latent until the cells are reactivated
by antigen. These memory cells may survive many years
and constitute a reservoir that is very important in drug-
based therapy.
 During this period, the virus disseminates to other regions
including to lymphoid and nervous tissue. This is the most
infectious
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 A latent reservoir
 As a result of the strong immune defence, the number of
viral particles in the blood stream declines and the patient
enters clinical latency.
 Little virus can now be found in the bloodstream or in
peripheral blood lymphocytes and, initially, the number of
blood CD4+ cells is only slightly decreased.
 Nevertheless, the virus persists elsewhere, particularly in
lymph nodes and here viral replication continues
 Although the number of HIV particles in the bloodstream
is much reduced during clinical latency, the virus is
detectable.
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 After the initial peak of virus, the virus reaches a "set
point" during latency. This set point predicts the time of
onset of clinical disease.
 With less than 1000 copies/ml of blood, disease will
probably occur with a latency period of more than 10
years. With less than 200 copies/ml, disease does not
appear to occur at all.
 Most patients with more than 100,000 copies per ml, lose
their CD4+ cells more rapidly and progress to AIDS
before 10 years.
 Most untreated patients have between 10,000 and 100,000
copies per ml in the clinical latency phase.
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 Loss of CD4+ cells and collapse of the immune response
 One reason that the immune system fails to control HIV infection is
that the CD4+ T helper cells are the target of the virus. Also
follicular dendritic cells can be infected with HIV and these also
diminish in number over time.
 Moreover, dendritic cells present antigen to CD4+ cells and may
bring the virus into contact with these cells at the time that they are
stimulated to proliferate by antigen.
During the course of infection, there is a profound loss of
the specific immune response to HIV because:
1. responding CD4+ cells become infected. The cells that
proliferate to respond to the virus are infected and killed by it

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2. epitope variation can lead to escape of HIV from the immune
response
3. activated CD4+ T cells are susceptible to apoptosis.
Spontaneous apoptosis of uninfected CD4+ and CD8+ T cells
occurs in HIV-infected patients.
4. the number of follicular dendritic cells falls over time, resulting in
diminished capacity to stimulate CD4+ cells
 There is thus a decline of CD4+ cells with especially a loss of those
specific to HIV. This occurs from the very beginning of infection
and is permanent (unless chemotherapy intervenes). Near the end
stage of AIDS, CD8+ cells also decline precipitously.
 During the course of HIV infection, most CD4+ cells are never
actually infected by the virus but die from some other means.
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 Onset of disease - AIDS
 The period of clinical latency varies in length from as little
as 1 to 2 years to more than 15 years. Onset of AIDS is
rare in less than 3 years except in children.
 Eventually, the virus can no longer be controlled as helper
CD4+ (T4) cells are destroyed. Ironically, the killer cells
needed to control HIV also damage the helper T cells that
they need to function efficiently.
 With the lack of CD4+ cells, new cytotoxic T cell
responses cannot occur as helper cells are lacking and such
new responses are required as the virus mutates. As the
CD4+ cells fall below 200 per cu mm, virus titers rise
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 It is the loss of immune competence that enables normally benign
opportunistic parasites such as viruses, fungi or protozoa to cause
infections
 Once AIDS develops, patients rarely survive more than two years
without chemotherapeutic intervention.
 There is considerable variability at this stage. Some patients with
clinical AIDS do survive for several years while others who appear
relatively healthy can suddenly succumb to a major opportunistic
infection.
 It is the onset of HIV-associated cancers and opportunistic
infections that defines AIDS proper. At this stage, also, syncytium-
inducing HIV appear in many (about half) AIDS patients. These are
more CD4+ cell tropic than the initially infecting HIV and this
contributes to the rapid loss of CD4+ cells in later stages of the
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 HERPES VIRUSES

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INTRODUCTION
 Herpes viruses are a leading cause of human viral disease,
second only to influenza and cold viruses. They are capable
of causing overt disease or remaining silent for many years
only to be reactivated, for example as shingles.
 The name herpes comes from the Latin herpes which, in
turn, comes from the Greek word herpein which means to
creep. This reflects the creeping or spreading nature of the
skin lesions caused by many herpes virus types.
 There are at least 25 viruses in the family Herpesviridae.
 Eight or more herpes virus types are known to infect man
frequently.
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HERPES VIRUS TYPES THAT INFECT HUMANS
 Herpes simplex virus Type 1 (HSV-1)
 Herpes simplex virus Type 2 (HSV-2)
 Epstein Barr virus (EBV)
 Cytomegalovirus (CMV)
 Varicella Zoster Virus (VZV)
 Human herpes virus 6 (exanthum subitum or roseola
infantum)
 Human herpes virus 8 (Kaposi's sarcoma-associate herpes
virus)

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 Once a patient has become infected by herpes virus, the
infection remains for life. The initial infection may be
followed by latency with subsequent reactivation.
 Herpes viruses infect most of the human population and
persons living past middle age usually have antibodies to
most of the above herpes viruses with the exception of
HHV-8.

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 HERPES SIMPLEX VIRUS (HSV)
 These are very large viruses.
 There are two types, HSV-1 and HSV-2 with very similar
characteristics
Pathogenesis
 The hallmark of herpes infection is the ability to infect
epithelial mucosal cells or lymphocytes. The virus then
travels up peripheral nerves to a nucleated neurone where
it may stay for years followed by reactivation.
 A reddened area gives rise to a macula which crusts to
form a papula. The fluid in this blister is full of virus. As
longjimmy
as ongori
the2013
virus is kept moist it can remain infectious
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 Herpes simplex 1 and 2 can infect both humans and other animals but
only humans show symptoms of disease.
 HSV-1 and HSV-2 first infect cells of the mucoepithelia or enter through
wounds. They then frequently set up latent infections in neuronal cells.
 Both types of HSV can also persistently infect macrophages and
lymphocytes.
 Once epithelial cells are infected, there is replication of the virus around
the lesion and entry into the innervating neurone. The virus travels along
the neurone (by a process called retrograde transport) to the ganglion.
 In the case of herpes infections of the oral mucosa, the virus goes to the
trigeminal ganglia whereas infections of the genital mucosa lead the virus
entering the sacral ganglia.
 Vesicles containing infectious virus are formed on the mucosa and the
virus spreads. The vesicle heals and there is usually no scar as a result.
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 Epidemiology
 HSV 1 and 2 infections are life-long and although latency is soon set up,
the infected patient can infect others as a result of recurrence.
 The virus is found in the lesions on the skin but can also be present in a
variety of body fluids including saliva and vaginal secretions.
 HSV-1 is usually spread mouth to mouth (kissing or the use of utensils
contaminated with saliva) or by transfer of infectious virus to the hands
after which the virus may enter the body via any wound or through the
eyes. A large proportion of the population has evidence of HSV-1
infection as judged by antibodies.
 As a result of poor hygiene in underdeveloped countries, HSV-1
antibodies are found in more than 90% of children.
 HSV-2 is normally spread sexually and is found in the anus, rectum and
upper alimentary tract as well as the genital area.
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 In addition an infant can be infected at birth by a genitally-
infected mother. The infant can also be infected in utero if
the mother's infection spreads.
 Anyone who comes in contact with fluid containing
infectious virus is at risk.
 As might be expected, HSV-2 infections are more
prevalent later in life as the number of sexual contacts
increases. Thus, the lowest rates of infection are found in
children and the highest rates in prostitutes among whom
as many as 80% are infected with HSV-2.

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 Diseases caused by Herpes Simplex Viruses
 Herpes simplex 1 and 2 are frequently benign but can also cause
severe disease. In each case, the initial lesion looks the same. A clear
vesicle containing infectious virus with a base of red (erythomatous)
lesion at the base of the vesicle. From this pus-containing
(pustular), encrusted lesions and ulcers may develop.
1. Oral herpes - Cold sores - Can be the result of an HSV-1 or an
HSV-
2 infection. Because of the association of HSV-2 with sexual
transmission,
infections in children are usually the result of HSV-1
2. Herpes keratitis - This is an infection of the eye and is primarily
caused by HSV-1. It can be recurrent and may lead to blindness.
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 3.Herpes whitlow - disease of persons who come in contact with
herpes-infected body secretions can be caused by either type of HSV
and enters the body via small wounds on the hands. It can also be
caused by transfer of HSV-2 from genitals to the hands.
4. Herpes gladiatorum
5. Eczema herpeticum
6. Genital herpes - Genital herpes is usually the result of HSV-2
with about 10% of cases being the result of HSV-1. Primary infection
is often asymptomatic but many painful lesions can develop on the
glans or shaft of the penis in men and on the vulva, vagina, cervix and
perianal region of women.

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 Secondary episodes of genital herpes, which occur as a
result of reactivation of virus in the sacral ganglion, are
frequently less severe (and last a shorter time) than the
first episode.
 Whether there is an apparent active disease or not, an
infected patient remains infectious without overt
symptoms. Clearly, these persons are very important in the
spread of herpes infection.
7. HSV proctitis - This is an inflammation of the rectum
and the anus

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8. HSV Encephalitis - This is usually the result of an
HSV-1 infection and is the most common sporadic viral
encephalitis.
9. HSV Meningitis - This is the result of an HSV-2
infection.
10.HSV infection of neonates
This results from HSV-2 and is often fatal, although such
infections are rare. Infection is especially possible if the
mother is shedding virus at the time of delivery.

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Diagnosis of HSV Infections
 Cells may be obtained from the base of the lesion (called a
Tzank smear) and histochemistry performed.
 The cells can also be stained with specific antibodies in an
immunofluorescence test
 It is also possible to detect viral DNA by in situ
hybridization
 Virus can be isolated from biopsy specimens, that is from
the lesions, and grown on tissue culture cells

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HSV chemotherapy
 Acycloguanosine (acyclovir), famciclovir and valacyclovir.
It should be noted that these drugs act against the
replicating virus and therefore they are ineffective against
latent virus.
 Since once the virus infects, the patient has it for life, the
best option is to avoid infection by not coming in contact
with the virus.
 Patients with genital herpes should avoid intercourse
when they have the prodromal itching symptoms or an
active lesion.
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 VARICELLA-ZOSTER VIRUS (ALSO KNOWN AS HERPES
ZOSTER VIRUS, HUMAN HERPES VIRUS-3)
 Zoster means girdle from the characteristic rash that forms a belt around
the thorax in many patients. The structure of Varicella virus is very
similar to Herpes Simplex virus
 Diseases caused by Varicella-Zoster virus
 This virus causes two major diseases, chicken-pox (Varicella), usually in
childhood, and shingles, later in life. Shingles (Zoster) is a reactivation of
an earlier varicella infection.
Chicken Pox
 This virus is highly infectious and even if we do not remember getting
it, more than 90% of the population antibodies against varicella proteins.
In the household of an infected patient, 90% of contacts who have
hitherto not had the disease will get it (unless vaccinated). It is spread by
respiratory aerosols
jimmy ongori 2013 or direct contact with skin lesions.
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Shingles
 After the infectious period, the virus may migrate to the
ganglia
 The virus may then be reactivated under stress or with
immune suppression. This usually occurs later in life.
 The skin lesions are different from those in chicken pox,
being restricted to small areas of the skin, usually in the
thorax
 Patients with AIDS often exhibit multi-dermatomal
recurrence of varicella infection. There is also a chronic
verricous form in some AIDS patients.
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 EPSTEIN- BARR VIRUS
 Epstein-Barr virus is the causative agent of Burkitt's lymphoma in
Africa, nasal pharyngeal carcinoma in the orient and infectious
mononucleosis in the west.
Burkitt's lymphoma
 The association between EBV and Burkitt's lymphoma has long
been established. This is a tumor of the jaw and face found in
children. The tumor cells show evidence of EBV DNA and tumor
antigens and patients show a much higher level of anti-EBV
antibodies than other members of the population.
 This lymphoma is endemic in equatorial Africa . Why this is so is
unclear but there is probably a genetic reason possibly involving an
association with malaria. Persons who are resistant to malaria
appear toongori
jimmy be2013
susceptible to progression to the lymphoma.
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 Nasopharyngeal cancer - This disease is also associated
with EBV. There may be a genetic predisposition to the
development of EBV cancers
 Oral hairy leukoplakia - This EBV-associated disease
results in lesions in the mouth and has increased in
frequency recently as it is an opportunistic infection of
HIV-infected patients
 Infectious mononucleosis

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 CYTOMEGALOVIRUS
Cytomegalovirus has the largest genome of all herpes viruses and appears only to
replicate in human cells. Its name
derives form the fact that, like other herpes viruses, it can form multinucleated
cells (syncytia) with characteristically
staining inclusions.
Transmission
 Cytomegalovirus infection is found in significant proportion of the population.
As with Epstein-Barr virus (also spread in saliva), seropositivity increases with
age.
 The virus is spread in most secretions, particularly saliva, urine, vaginal
secretions and semen
 Cytomegalovirus infection is therefore sexually transmitted. It can also spread
to a fetus in a pregnant woman and to the newborn via lactation,
 In the hospital, the virus can also be spread via blood transfusions and
transplants.
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 Pathogenesis
 Cytomegalovirus causes no symptoms in children and at most mild
disease in adults
Congenital disease
 During a primary infection of the mother, the virus can spread via
the placenta to the fetus and congenital abnormalities can occur; in
fact, this virus is the most common viral cause of congenital disease.
Disease in immunosuppressed patients
 In patients who have received an organ transplant or have an
immunosuppressive disease (e.g. AIDS), cytomegalovirus can be a
major problem. Particularly important is cytomegalovirus-retinitis
in the eye which occurs in up to 15% of all AIDS patients. In
addition, interstitial pneumonia, colitis, esophagitis and encephalitis
are seen in some patients.
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 HEPATITIS VIRUSES

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 Several diseases of the liver, collectively known as hepatitis, are
caused by viruses. The viruses involved, five of which have been
reasonably well characterized, come from a wide range of virus
families.
 Hepatitis A virus is a picornavirus, a small single strand RNA virus;
 Hepatitis B virus belongs to the hepadnavirus family of double
stranded DNA viruses;
 Hepatitis C virus is a flavivirus, a single stand RNA virus;
 Hepatitis E, also an RNA virus, is similar to a calicivirus.
 Hepatitis D which is also known as Delta agent is a circular RNA
that is more similar to a plant a viroid than a complete virus.

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HEPATITIS A VIRUS
 This picornavirus is the causative agent of infectious hepatitis

HEPATITIS B VIRUS
 Human hepatitis B virus belongs to the hepadnavirus family and
causes serum hepatitis.
 It infects humans and chimpanzees
 HBV is a DNA virus and is enveloped. The DNA is only partly
double stranded
 It is heat- and pH-resistant.

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 Depending on the patient’s immune response, infection by
HBV can be asymptomatic, chronic or acute.
 Humans are the only reservoir for HBV. The virus is spread
via contact with body fluids – blood, semen and various
secretions eg vaginal fluids, menstrual blood, saliva and
milk.
 Although injection of blood is the most common route of
infection, the virus can also be contracted via sexual
intercourse (particularly male to male) and perinatally.
 About 5% of people infected by HBV get a chronic
infection. Up to one quarter of these chronically-infected
patients will die of some form of liver disease.
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Pathology
 HBV enters the body in the bloodstream and targets hepatocytes
 The incubation period is 60-90 days
 The first sign of infection is the characteristic appearance of HBsAg
in infected cells.
 The symptoms are immune-mediated, resulting from inflammation
and cell-mediated (cytotoxic T cell) responses to HBsAg on the
surface of hepatocytes.
 Chronic HBV infection can lead to chronic hepatitis. This leads to
cirrhosis of the liver in up to a quarter of patients within five years.
Of these patients, up to one quarter will develop hepatocellular
carcinoma or liver failure. Both of these are fatal in the absence of a
liver transplant.
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Diagnosis
 Serum hepatitis is usually first diagnosed from the clinical
symptoms. Liver enzymes are also detected in the bloodstream
during the symptomatic phase.
 An acute infection can be distinguished from a chronic infection by
the presence of antibodies (IgM) against HBcAg. Tests that detect
HBsAg and HBcAg
 The presence of HBeAg is the best marker for infectious virus.
 HBV can also be detected in the laboratory by
immunohistochemistry (fi

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 Carcinogenesis
 It is clear that individuals who are HBsAg positive are at a much
higher risk of hepatocellular carcinoma than those who are
negative.
 In patients with chronic hepatitis, there is destruction of
hepatocytes as a result of the immune response to the virus. This
results in regeneration (by cell division) of liver cells that may
ultimately cause the cancer.
 Although the virus does not integrate during the course of normal
replication, parts of the HBV genome are found integrated into the
DNA of hepatocellular carcinoma patients.
 Hepatocellular carcinoma takes many years to develop

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 HEPATITIS C VIRUS
 Hepatitis C is a flavivirus that causes non-A, non-B hepatitis

jimmy ongori 2013

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  MEASLES (RUBEOLA)

The word measles is derived from the German word for blister.
 Before the advent of the current measles vaccine, there were
about 500,000 cases of measles in the United States per year;
almost everyone got the measles. But since 1963, the number has
fallen precipitously (figure 1B) with a low of only 86 cases in
2001, all of which seem to be imported. In the less developed
world, measles still takes its toll with an estimated 30 million
illnesses and 770,000 measles-caused deaths in 2000 of which
58% were in Africa

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  PATHOGENESIS AND DISEASE (figure 2)
Infection is via an aerosol route and the virus is very contagious. It replicates initially in the upper/lower respiratory tract, followed by
replication in lymphoid tissues leading to viremia and growth in a variety of epithelial sites. The disease develops 1 - 2 weeks after infection.
 Uncomplicated disease is characterized by the following:
 Fever of 101 degrees Fahrenheit (38.3 C) or above
 Respiratory tract symptoms: running nose (coryza) and cough
 Conjunctivitis (table 2)
 Koplik's spots on mucosal membranes (table 2) - small (1 - 3mm), irregular, bright red spots, with bluish-white speck at center. The patient
may get an enormous number and red areas may become confluent (see below).
 Maculopapular rash which extends from face to the extremities. This seems to be associated with T-cells targeting infected endothelial cells in
small blood vessels (table 2) (see below).

 The infection is prostrating but recovery is usually rapid. The peak of infectiousness is before the onset of obvious symptoms (Koplik’s spots,
rash). Note some virus shedding occurs during the disease phase, so spread of the virus to other individuals can be somewhat reduced by
minimizing contact with others.
 The cell mediated response is important since patients with agamma-globulinemia recover normally. Measles tends to be more severe in
adults and the very young (under 5 years of age) and is less severe in older children and teenagers.
 Complications of measles
 If a patient has an impaired cell-mediated immune response, there is continued growth of the virus in the lungs leading to giant cell
pneumonia (such patients may not have a rash). This is rare, but often fatal. The reason for the giant cells is that, since F protein can function
at physiological pH, it can facilitate cell-cell fusion.
 Since virus grows in epithelia of the nasopharynx, middle ear and lung, all of these sites may then be susceptible to secondary bacterial
infection. Otitis media and bacterial pneumonia are quite common.
 The outcome of the disease is affected by the nourishment of the patient and access to medical care. Measles is still a major killer in
underdeveloped countries and several studies in areas with severe vitamin A deficiency problems have found that vitamin A treatment of
children with measles has resulted in reduction in morbidity and mortality. Pneumonia accounts for 60% of deaths from measles.
 One in 1000 cases may get encephalitis a few days after the rash disappears. Most patients (90%) survive encephalitis but there may be
complications such as deafness, seizures and mental disorders.

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 jimmy ongori 2013
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 jimmy ongori 2013
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 jimmy ongori 2013
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 jimmy ongori 2013
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 HUMAN IMMUNODEFICIENCY
VIRUS AND AIDS

562 jimmy ongori 2013 03/11/2013 21:19

 Human Immunodeficiency Virus:
Historical Background
1981 Doctors in US recognized PCP in homosexual males, a
condition previously unreported in healthy adults
• Later recognized that all these patients were
immunosuppressed
1983/4 Scientist described the cause of this acquired immunodeficiency
syndrome (AIDS) as a retrovirus
– Lymphadenopathy Associated Virus (LAV)
– AIDs Associated Retrovirus (ARV)
– Human T- lymphotrophic Virus Ш (HTLV-Ш)
1984 First case described In Kenya
1986 Human Immunodeficiency Virus (HIV) accepted as international
designation for the retrovirus in a WHO consultative meeting

563 jimmy ongori 2013 03/11/2013 21:19

 Human Immunodeficiency Virus:
Historical Background (cont.)
1996 ARVs available in the world

1997 ARVs available in Kenya private sector

2003 ARVs available in Kenya public sector

2005 54,000 patients on ART

2010 Approx.426,870 patients on ART

564 jimmy ongori 2013 03/11/2013 21:19

HIV VIRUS
 AIDS is caused by Human Immunodeficiency Virus
(HIV), A retrovirus from the Lentivirus family.
 The primary targets of HIV are activated CD4+ T4
helper lymphocytes but the virus can also infect several
other cell types including macrophages.
 Loss of T4 helper lymphocytes leads
to immunosuppression in the patient and the
consequent fatal opportunistic infections.
 Genetic material consists of a single-stranded
ribonucleic acid (RNA)
565 jimmy ongori 2013 03/11/2013 21:19
The Biology Of The HIV
 HIV is a lentivirus, a class of retrovirus.
 The name lentivirus means slow virus, so called because these
viruses take a long time to cause overt disease.
 Most lentiviruses target cells of the immune system and thus
disease is often manifested as immunodeficiency.
 There are five known serogroups of lentivirus that infect
primates, sheep and goats, horses, cats, and cattle.
 There are two types of HIV: HIV-1 and HIV-2. These cause
clinically indistinguishable disease, although the time to
disease onset is longer for HIV-2. The worldwide epidemic
of HIV and AIDS is caused by HIV-1 while HIV-2 is mostly
restricted to west Africa.
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 HIV – 1
 Is found worldwide
 Is the main cause of the worldwide pandemic
 HIV – 2
 Is mainly found in West Africa, Mozambique and
Angola.
 Causes a similar illness to HIV – 1
 Less efficiently transmissible rarely causing vertical
transmission
 Less aggressive with slower disease progression

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HIV-1 Subtypes
 HIV-1 has many subtypes: A-K
 A-E are the predominant subtypes
 A: W. Africa, E. Africa, Central Africa East
Europe & Middle East
 B: N. America, Europe, Middle East, E. Asia, Latin
America
 C: S. Africa, S. Asia, Ethiopia
 D: E. Africa
 E: S. E. Asia

568 jimmy ongori 2013 03/11/2013 21:19

 Unlike other retroviruses, which bud from the
infected cell, HIV can lie dormant in the proviral
form within a cell for many years, especially in
resting (memory) CD4+ T lymphocytes, and may set
up a lifelong infection.
 When these cells become reactivated, viral
production occurs again and ultimately destroys the
cell. Although HIV may disappear from the cells of
the circulation, replication and budding continue to
occur in other tissues in the absence of
chemotherapy
569
jimmy ongori 2013 03/11/2013 21:19
Structure Of HIV
 Has an outer double
lipid membrane,
(derived from the host
membrane).
 The lipid membrane is

lined by a matrix
protein.
 The lipid membrane is

studded with the surface

glycoprotein (gp) 120
and the transmembrane
gp 41 protein.
 These glycoprotein
spikes surround the
cone-shaped
570
protein
jimmy ongori 2013 03/11/2013 21:19
core.
HIV Structure
Viral Enzymes
 Most important: Reverse Transcriptase (RT), Protease and
Integrase.
 RT converts viral single-stranded RNA into a double
stranded deoxyribonucleic acid (DNA).
 DNA is incorporated into host nucleus as the proviral
DNA.
 Integrase facilitates integration of the DNA into the host’s
chromosomal DNA.
 Protease enzyme splits generated macro-proteins into
smaller viral proteins (core, envelope & regulatory
proteins and enzymes) which go into forming new viral
particles.
571 jimmy ongori 2013 03/11/2013 21:19
HIV Life Cycle
 Binding, Fusion
and Entry
 Transcription
 Integration &
Replication
 Budding
 Maturation

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BINDING:
 For successful entry into cells the HIV envelope
glycoprotein GP 120 binds to the host receptor CD4
molecule
FUSION and ENTRY:
 Viral binding to host cell triggers fusion of the viral
and host cell membranes
 Mediated by gp41
 Allows entry of virus core into host cell cytoplasm
 Core protein dissolved by host enzymes releasing
viral RNA and enzymes
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INTEGRATION
 Reverse transcriptase converts the viral RNA into a DNA
molecule
 The DNA enters the host cell nucleus
 Integrase catalyses the process of integration of the viral
DNA into the host cell’s DNA
REPLICATION
 Integrated viral DNA turns the host cell into a "factory"
for manufacturing more virus.
 Viral proteins are produced as a single multi-protein
molecule
 Viral
574 jimmy proteins
ongori 2013 cleaved by protease enzyme 03/11/2013 21:19
Budding and Maturation:
 Viral proteins together with RNA gather at the membrane
of the CD4+ cells
 Viral particles are formed which bud off the cell and enter
the bloodstream
 The CD4 cells are often destroyed by HIV virus infection
and replication resulting in profound immunodeficiency.

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 HIV LIFE CYCLE: Enzymes
 Transcription:
 Activation of host cell
Fusion & entry results in transcription
of viral DNA into
mRNA.
 mRNA translated into
viral precursor
proteins
 Reverse  Assembly &
Transcription Budding
 The viral enzyme  Viral precursor proteins
reverse processed by protease
transcriptase enzyme into usable forms
 Proteins assembled with
converts the single RNA to form viral
stranded viral RNA particles which then bud
into double strand Integration:
DNA The viral enzyme
integrase inserts the
viral DNA (viral genetic
material) into the host
576 jimmy ongori 2013 03/11/2013 21:19
DNA.
  To show the video clips of viral replication and targets of
ARVs

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 Global summary of the AIDS epidemic 
2009

Number of people Total

33.3 million [31.4 million–35.3
living with HIV Adults
million]
Women 30.8 million [29.2 million–32.6
Children (<15 years)
million]
15.9 million [14.8 million–17.2
million]
People newly Total 2.5 million [1.6 million–3.4 million]
infected Adults
with HIV in 2009 Children (<15 years)
2.6 million [2.3 million–2.8 million]
2.2 million [2.0 million–2.4 million]
AIDS deaths in 2009 Total 370 000 [230 000–510 000]
Adults
Children (<15 years)
1.8 million [1.6 million–2.1 million]
1.6 million [1.4 million–1.8 million]
578 jimmy ongori 2013 260 000 [150 000–360 000] 21:19
03/11/2013
 Impact of HIV in Kenya
 National HIV prevalence is 6.3% based on Kenya
Demographic Health Survey (KDHS 2008/9)

 1.45 m Kenyans estimated to be HIV positive and

about 650, 000 need antiretroviral therapy (ART)

 Since 1984 Kenya has experienced a negative impact

on all sectors of society as a result of AIDS
epidemic.

 Reversed previous health gains: life expectancy

reduced
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Modes of Transmission
Sexual contact
 In Africa mainly heterosexual
 Homosexual
 Non-consensual sexual exposure (rape)
Parenteral
 Transfusion of infected blood or blood products
 Exposure to infected blood or body fluids through
contaminated sharps, needle-sharing or needle stick
accidents
 Donated organs, Traditional procedures
Perinatal
 Transplacental, during labor/delivery and breastfeeding
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 Percent infection by transmission route….

Transmission route %

Sexual intercourse 70-80

Mother-to-child-transmission 5-10

Blood transfusion 3-5

Injecting drug use 5-10

Health care – e.g. needle stick <0.01

injury

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Biological Factors Influencing HIV Transmission
 Disease status of source patient
 Degree of immunosuppression and viral load - High risk
during primary infection and late disease when viral load
high
 Presence of untreated STIs in source and person at
risk
 Both ulcerative and non ulcerative STIs important
cofactors - Related to high viral load in genital secretions
during STIs and the disturbance of the genital mucosa
 A major reason for high prevalence in Sub Saharan Africa

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 Circumcision status
 Uncircumcised men 2x as likely to acquire HIV
infection than circumcised. Also more likely to
acquire STIs

 Gender differences in susceptibility

 Female genital anatomy presents a larger surface area
with more of the cells that HIV requires to gain entry
 (Socio-cultural factors)

 Host genetic differences

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Socio-economic Factors
 Social Mobility
 HIV/AIDS follows routes of commerce
 Partners living apart
 Stigma and Denial
 Denial and silence is the norm
 Stigma prevents acknowledgment of problem and
care-seeking
 People in Conflict
 Context of war and struggle of power spreads AIDS
 Cultural Factors
584 jimmy ongori 2013 03/11/2013 21:19
 Socio-economic Factors (cont’d)
• Gender
– In many cultures it is accepted for men to have many
sexual relationships
– Many women unable to negotiate condom use

• Poverty
– Lack of information needed to understand and
prevent HIV
• Drug Use and Alcohol Consumption
– Impaired judgment
– Sharing of needles and equipment
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 Behavioral Factors
 Multiple sexual partners
 Unprotected sexual intercourse
 Large age difference between sexual partners

Factors not associated with risk of transmission

 Insect bites
 Saliva (kissing)
 Sneezing or coughing
 Skin contact (e.g. hugging)
 Shared use of facilities (e.g. toilets)

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 Cells of the immune system
 Responsible for protecting the body from invading
foreign bodies - Found in blood and tissues
 In blood mostly are white blood cells (WBC)
 Macrophages clearing the body of infected, old or
damaged cells
 Neutrophils attack bacteria
 Eosinophils attack worms (and mediate allergies)
 B-lymphocytes make antibodies
 T-lymphocytes
 Attack viruses, fungi and some bacteria like
mycobacteria
 T helper (CD4) cells assist in function of other
immune cells
587 jimmy
CD8 ongori 2013
or T killer cells are able to destroy infected cells
03/11/2013 21:19
 How HIV Affects Immune System
 HIV attaches to cells of the immune system through special
surface markers called CD4 receptors
 The following immune cells have CD4 receptors
 T-Lymphocytes – CD4+ Cells
 Macrophages
 Monocytes
 Dendritic cells
 HIV infection of CD4 cells causes cell dysfunction and death
 The hallmark of HIV/AIDS is a profound immunodeficiency as a
result depletion of CD4+ T lymphocytes.
 The CD4+ T cell depletion is two fold
 Reduction in numbers
 Impairment in function
588 jimmy ongori 2013 03/11/2013 21:19
 Reduction in the CD4 cell number and the effects
on their function reduces the capacity of the body
to fight infectious diseases.
 Individuals with HIV infection are therefore
increasingly susceptible to many infections
especially at later stages of HIV infection

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Primary HIV Infection
 On exposure, there is a 2-4 week period of intense viral
replication and widespread dissemination of virus
characterized by
 High plasma viral load (RNA)
 Rapid decline in CD4 count
 In some cases an acute illness occurs
 Lasts from 1-2 weeks, but it is rarely diagnosed
 Symptom resolution with reduction in plasma
viremia due to development of an immune
response and antibodies to the virus

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Asymptomatic Disease (Latency)
 Patients then enter a stage of asymptomatic disease
phase lasting on average 2-10 years (clinical
latency)
 Characterized by gradual decline in CD4 count
 Rate depends on viral load
 Host genetic/immunological or viral factors may
be involved

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Symptomatic Disease and AIDS
 Viral load continues to rise causing
 Increased demands on immune system as production of
CD4 cells cannot match destruction
 Increased susceptibility to common infections (URTI,
pneumonia, skin etc)
 Late-stage disease is characterized by a CD4 count
<200cells/mm3 and the development of opportunistic
infections, selected tumors, wasting, and neurological
complications).
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Opportunistic Infections
 The predominant causes of morbidity and mortality
among patients with late-stage HIV infection are
opportunistic infections, ie, severe infections induced
by agents that rarely cause serious disease in immune-
competent individuals.
 The most common opportunistic infections in untreated
AIDS patients include the following:
1.Protozoa: Toxoplasma gondii, Isospora belli,
Cryptosporidium species.
2. Fungi: Candida albicans, Cryptococcus neoformans,
Coccidioides immitis, Histoplasma capsulatum, Pneumocystis
jiroveci. 03/11/2013 21:19
593 jimmy ongori 2013
Opportunistic Infections
3. Bacteria: Mycobacterium avium-intracellulare,
Mycobacterium tuberculosis, Listeria monocytogenes, Nocardia
asteroides, Salmonella species, Streptococcus species.
4. Viruses: Cytomegalovirus, herpes simplex virus,
varicella-zoster virus, adenovirus, polyomavirus, JC
virus, hepatitis B virus, hepatitis C virus.
 Herpesvirus infections are common in AIDS patients,
and multiple herpesviruses are frequently detected
being shed in saliva. Cytomegalovirus retinitis is the
most common severe ocular complication of AIDS.
 Cancer
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 Laboratory diagnosis
 Serological methods: antibody/antigen based
test
 Enzyme Immunosorbent Assay (ELISA)

 Western Blot
 Viral detection methods

 PCR

 Culture: Rarely used

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 Serological methods
 Common and widely used tests

 Based on the principal of antigen antibody reaction

 Antibody takes some time to be produced after an

infection

 Therefore all these tests have a problem of making a

diagnosis during the period immediately after infection
with HIV prior to the appearance of detectable antibodies

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 Rapid Tests
 These are ELISA tests

 Can be performed in less than 20 minutes

 therefore also referred as ―Simple/Rapid‖ (S/R) assays

 Can be performed easily without instruments

 A positive result is indicated by the appearance of a

colored dot or line

 Examples – Bioline, Determine, Unigold and Oraquick.

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597
 Limitations of the HIV test

 A negative result doesn’t eliminate the possibility of HIV

1/HIV 2 infection.

 The specimen may contain low levels of antibodies to

HIV 1/HIV 2.

 Diagnosis is based on more than one kit

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 1. PICORNAVIRUSES
 Picornaviruses are the small RNA
 Have a naked nucleocapsid that is about 30mm in diameter.
 Pico means small, hence small RNA viruses or
 There are nine genera within thePicornaviridae. Five of
these infect humans:
 Enteroviruses
 Rhinoviruses
 Hepatoviruses
 Parechoviruses
 Kobuviruses

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 Enterovirus
Polio Diseases of the human (and
Coxsackie A and B other) alimentary tract (e.g.
Echo polio virus)
Other enteroviruses
Disease of the nasopharyngeal
Rhinovirus
region (e.g. common cold virus)
Hepatovirus Human hepatitis virus A
Formerly echoviruses 22 and 23.
Parechovirus Disease of alimentary and
respiratory tract
Kobuvirus Aichi virus is the type species
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ENTEROVIRUSES
 Spread via the fecal-oral route. The ingested
viruses infect cells of the oro-pharyngeal mucosa
and lymphoid tissue (tonsils) where they are
replicated and shed into the alimentary tract
 Pass into the intestine and set up further infections
in the intestinal mucosa. The virus also infects the
lymphoid tissue (Peyer's patches)
 The virus replicates and are shed into the feces,
often for months after the primary infection
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601
 POLIOVIRUS
 Poliomyelitis means inflammation of the gray (poliós)
spinal cord (myelós). It is also known as infantile
paralysis.
 First recorded case of poliomyelitis comes from an
Egypt (1580-1350 BCE)
 There are three serotypes of polio virus. Most disease
results from type 1 polio virus.
 Since the disease is spread by fecal contamination,
infections are more common where unsanitary
conditions prevail but many children in these areas have
asymptomatic infections that lead to life-long immunity.
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602 jimmy ongori 2013
 Asymptomatic polio infection
 In more than 90% of the cases, infection is asymptomatic.
This occurs when the replication of the virus is restricted to
the GIT
 Why many polio infections are asymptomatic may be due to;
the size of the innoculum of the virus, the size of the resulting
viremia, the virulence of the infecting virus, and the presence
of circulating antibodies.
Abortive poliomyelitis (minor illness)
 The first symptomatic result of polio infection is febrile
disease and occurs in the first week of infection. The patient
may exhibit a general malaise which may be accompanied by
vomiting, a headache and sore throat. This is abortive
poliomyelitis and occurs in about 5% of infected individuals
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jimmy ongori 2013
 Non-paralytic poliomyelitis
 Three or four days later a stiff neck and vomiting, as a
result of muscle spasms, may occur in about 2% of
patients. This is similar to aseptic meningitis. The virus has
now progressed to the brain and infected the meninges.
Paralytic polio
 About 4 days after the end of the first minor symptoms,
the virus has spread from the blood to the anterior horn
cells of the spinal cord and to the motor cortex of the
brain. The degree of paralysis depends on the which
neurons are affected and the amount of damage that they
sustain. The disease is more pronounced in very young
and very old patients
604
jimmy ongori 2013 03/11/2013 21:19
 In spinal paralysis one or more limbs may be affected or
complete flaccid paralysis may occur.
 In bulbar paralysis cranial nerves and the respiratory center in
the medulla are affected leading to paralysis of neck and
respiratory muscles.
 The degree of paralysis may increase over a period of a few
days and may remain for life or there may be complete
recovery over period of 6 months to a few years.
Post-polio syndrome
 This afflicts victims of an earlier polio virus infection but the
virus is no longer present. It may occur many years after the
infection and involves loss of function in affected muscles,
perhaps as a result of further neuron loss.
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COXSACKIE VIRUSES
 Coxsackie type A usually is associated with surface rashes
(exanthems) while type B typically causes internal symptoms (eg
myocarditis) but both can also cause paralytic disease or mild
respiratory tract infection.
 Meningitis
 Enteroviruses are the major cause of viral meningitis. Both
Coxsackie virus A and B can cause aseptic meningitis which is so-
called because it is not of bacterial origin. Viral meningitis
typically involves a headache, stiff neck, fever and general malaise.
PREVENTION OF PICORNAVIRUS DISEASE
 Vaccination is the major means of control of this virus
 There are no vaccines for Coxsackie virus or other enteroviruses

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 RHINOVIRUSES
 Rhinoviruses are one of the families of viruses that can cause
the common cold although many other viruses can infect the
respiratory tract and cause cold-like symptoms.
 About one third of "colds" are caused by rhinovirus
infections. There are more than 100 serotypes explaining
why vaccines against rhinoviruses have proved difficult to
develop.
 Are spread by aerosols and infect the upper respiratory tract,
can also be spread by fomites such as hands and other forms
of direct contact.
 Are sensitive to temperature. Thus, they do not spread to the
lower respiratory tract since they replicate best at a few
degrees below normal body temperature. 03/11/2013 21:19
jimmy ongori 2013
 RHINOVIRUS DISEASE
 There are nearly 62 million cases of the common cold annually in the
US
 52.2 million of these cases affect Americans under age 17
 There are nearly 22 million school-loss days annually due to the
common cold
 There are approximately 45 million bed days annually associated with
the common cold
 Seventy-five percent of common colds suffered by children under 5
years are medically attended
Source: Vital and Health Statistics Series 10, No. 200
 The symptoms of a rhinovirus infection are well known: discharging or
blocked nasal passages often accompanied by sneezes, and perhaps a
sore throat. This typical "runny nose" (rhinorhea) may be accompanied
jimmy ongori 2013
608 by a general malaise, cough, sore throat etc. The characteristic
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