4.

ANTIBODIES

Definition

Structure

Types

Properties and Functions of Immunoglobulins

Production of Polyclonal and Monoclonal Antibodies

Applications
ILLNESS DISORDER

SICKNESS CONDITION

DISEASE SYNDROME
•A disease is a pathophysiological response to internal or external factors.

•A disorder is a disruption to regular bodily structure and function.

•A syndrome is a collection of signs and symptoms associated with a specific

health-related cause.

•A condition is an abnormal state of health that interferes with normal or

regular feelings of wellbeing.

 What are pathogens?
Classification
Culture A pathogen is an organism that causes disease.
Biochemical
characters
Antigenic structure
Pathogenicity
Pathogenesis
Clinical diagnosis
Laboratory diagnosis
Epidemiology
Prophylaxis
Chemotherapy
Classification
Biochemical tests Cultural characters
When grown on a variety of media,
microorganisms will exhibit
the difference in the microscopic
appearance of their broth. These
differences are called cultural
characteristics and are used as a basis for
separating microorganism into taxonomic
groups.
Antigen
“An antigen is a molecule that triggers the production of an antibody and causes an immune response.”
Antigens are large molecules of proteins, present on the surface of the pathogen- such as bacteria, fungi
viruses, and other foreign particles.

Structure of Antigens
The epitopes or antigenic determinants are the components of
antigen. Every antigen has several epitopes. An antibody has at least
two binding sites that can bind to specific epitopes on antigens.
The antigens combine with the antibody according to the lock and
key mechanism.
The ability of the body to act against the disease-causing agents and
antigens by the immune system is termed as the immunity. This
immunity may be either inborn or acquired from vaccinations.
✓ Pathogenicity is the quality or state of being
pathogenic, the potential ability to produce disease
whereas virulence is the disease producing power of
an organism, the degree of pathogenicity within a
group or species.
✓ A related term to pathogenicity is virulence, which
refers to the degree of pathogenicity of a particular
organism. virulence is a term that quantifies
pathogenicity.
✓ The host may be a particular animal, plant, fungal, or
another microbial species.

Pathogenesis : The Pathos (origination) and genesis

(development of a disease)
Clinical diagnosis
The process of identifying a disease,
condition, or injury based on the signs and
symptoms a patient is having and the
patient's health history and physical exam.
Laboratory diagnosis
A medical procedure that involves testing a sample of blood,
urine, or other substance from the body. Laboratory tests can
help determine a diagnosis, plan treatment, check to see if
treatment is working, or monitor the disease over time.
Epidemiology is the study and analysis of the distribution, patterns and determinants of health and disease
conditions in a defined population.
It is a cornerstone of public health, and shapes policy decisions and evidence-based practice by identifying risk
factors for disease and targets for preventive healthcare.
Prophylaxis Chemotherapy is a drug treatment that
Measures designed to preserve health (as of an individual uses powerful chemicals to kill fast-
or of society) and prevent the spread of disease growing cells in your body.

✓ Pre-exposure prophylaxis
✓ Post exposure prophylaxis
Bacterial Diseases Protozoan Diseases
1. Syphilis 1. Amoebiasis
2. Diphtheria 2. Malaria
3. Tetanus
4. Typhoid
5. Cholera
6. Tuberculosis

Viral Diseases Fungal Diseases

1. Rabies 1. Candidiasis
2. Hepatitis A,B 2. Cutaneous mycoses
3. HIV
Tetanus

Bacterial Disease

Clostridium tetani (C. tetani)

Tetanus notes: Dr Akshata G Athreya
Tetanus is an infectious disease characterized by increased muscle tone and spasms caused
by the release of tetanospasmin, a neurotoxin produced by C. tetani when it is inoculated
into humans.

Tetanus notes: Dr Akshata G Athreya

•Characterized by generalized rigidity and convulsive spasms

•First produced in animals in 1884

•Organism isolated in 1889

•Tetanus toxoid developed in 1924 and widely used during

World War II

Classification
Habitat of Clostridium tetani Kingdom Bacteria

Phylum Firmicutes
•Organisms of C. tetani are discovered in dirt, animal faeces, and
Class Clostridia
occasionally human excrement, in addition to inanimate things. Order Clostridiales
Family Clostridiaceae
Genus Clostridium
•In some conditions, the spores can persist for years and are Species tetani
Tetanus notes: Dr Akshata G Athreya
resistant to disinfectants and even 20 minutes of boiling water.
Morphology of Clostridium tetani

•4–8 m in length
•slender
•Bacillus
•obligate anaerobic
•Gram-positive
•The bacillus occurs in singles, occasionally in chains.
• consist of round, terminal, and bulging spores giving
drumstick appearance to the bacillus. The spores are rarely
seen even in clinical specimens from lesions or in culture
media.
•Except for type VI, all strains of C. tetani are motile due
to the presence of flagella. Bacilli of strain type VI lack
flagella and are therefore non-motile.
•Bacteria are encapsulated.
Tetanus notes: Dr Akshata G Athreya
 Culture
• CULTURE REQUIREMENTS OF CLOSTRIDIUM TETANI
• ⇒ Special requirements – no complex nutritional requirements and readily grow on NAM. Also,
it grows well in the media containing Blood or Serum, commonly Blood Agar medium &
Robertson Cooked meat broth medium is used for the cultivation of Clostridium tetani in
Laboratory.
• ⇒ Optimum temperature – 14 –43°C as per the strain, usually grown at 33–37°C in laboratories.
• ⇒ Optimum pH – 7.4 – 7.6
• ⇒ Oxygen requirements – obligate anaerobic bacterium i.e. can only grow in the absence of
oxygen (Strict anaerobe).
• ⇒ There are various culture media used for the cultivation of C. tetani in the laboratory and most
commonly the Sheep Blood Agar medium and CMB medium is used, the other media are as
follows:-
• Nutrient Agar medium
• Columbia Horse Blood Agar medium
• The liquid medium (Nutrient Broth medium, TSB (Tryptic soy broth medium, etc.).
Tetanus notes: Dr Akshata G Athreya
Cultural Stiff Sheep Blood Agar Nutrient Agar medium
Characteristics Medium (3% Agar) (NAM)
Shape Irregular Irregular
Size 0.5-1 mm Swarming growth
Elevation Slightly raised Flat
Color Grey Colorless – white
Surface Rough Swarming growth
spread throughout the
plate with rough surface
Structure Translucent –Opaque Transparent -
Translucent
Hemolysis Alpha hemolysis initially, -----
followed by beta - hemolysis

Tetanus notes: Dr Akshata G Athreya

 Biochemical characters

Fermentation of Maltose Negative (-ve)

Arabinose Negative (-ve) Mannitol Negative (-ve)
Cellobiose Negative (-ve) Mannose Negative (-ve)
DNase Positive (+ve) Raffinose Negative (-ve)
Rhamnose Negative (-ve)
Fructose Negative (-ve)
Ribose Negative (-ve)
Galactose Negative (-ve)
Salicin Negative (-ve)
Glucose Negative (-ve)
Sorbitol Negative (-ve)
Glycogen Negative (-ve) Starch Negative (-ve)
Inositol Negative (-ve) Sucrose Negative (-ve)
Inulin Negative (-ve) Trehalose Negative (-ve)
Lactose Negative (-ve) Xylose Negative (-ve)
Tetanus notes: Dr Akshata G Athreya
Basic Characteristics Properties (Clostridium tetani)
Capsule Negative (-ve)
Catalase Negative (-ve)
Flagella Peritrichous
Gas Positive (+ve)
Gelatin Hydrolysis Positive (+ve)

Gram Staining Gram-stain-positive

Growth in 20% Bile Negative (-ve)

Growth in 6.5% NaCl Negative (-ve)
H2S Positive (+ve)
Hemolysis (lysis of RBC) Positive (+ve), first alpha and then beta
Indole Variable
Methyl Red / Voges-Proskauer Negative (-ve)
Motility Positive (+ve)
Nitrate Reduction Negative (-ve)
Tetanus notes: Dr Akshata G Athreya
Spore Positive (+ve)
Antigenic/Virulence factors of Clostridium tetani and structure

The minimum lethal dose

(MLD) of a poison is :
10–6 mg for animals

130 ng for humans.

Birds and reptiles possess

exceptional resistance to the
poison.

Equine species are the most

vulnerable to the toxin,
followed in descending order
by guinea pigs, goats, and
rabbits… Humans.
Tetanus notes: Dr Akshata G Athreya
Tetanus notes: Dr Akshata G Athreya
❖ Pathogenicity can be defined as the capacity of a microbe to cause damage in a host while
virulence refers to the degree of damage caused by the microbe
❖ Pathogenicity is expressed by microbes using their virulence, or the degree of the microbe's
pathogenicity. Genetic, biochemical, and structural features that lead to the ability of the
pathogen to cause disease are known as its determinants of virulence.
❖ Tetanus toxin, a neurotoxin (tetanospasmin ), is the most important virulence factor that
plays a key role in the pathogenicity of tetanus . Confirmation of virulence by confirming
the production of tetanospasmin by infecting species forms the most important part in the
diagnosis of tetanus .
❖ Clostridium tetani is one of the four most well-known exotoxin-producing
pathogens within this category and the sole causative organism for the disease known as
tetanus. Tetanus notes: Dr Akshata G Athreya
Tetanus is caused by C. tetani spores entering the body. Pathogenesis

Under favourable conditions of anaerobiosis, the spores germinate into a vegetative state and then
create toxins such as tetanospasmin and tetanolysin.

Wounds with low oxidation–reduction potential, such as those with (a) dead devitalized tissue, (b) a
foreign body, or (c) an active infection, promote anaerobiosis in tissues.

Locally and peripherally at the myoneural junction of the nervous system, tetanospasmin is absorbed
and transported centripetally into CNS neurons.

•The heavy chain of the toxin is responsible for protein transport and particular binding to brain
cells.
•The light chain inhibits the release of Tetanus
GABA and glycine, two key inhibitory neurotransmitters.
notes: Dr Akshata G Athreya

Mechanism of Tetanus Toxin
Toxin binding cannot be reversed.
Lack of inhibitory signals to the motor neurons and
constant release of acetylcholine to the muscle fibers
leads to irreversible contraction of the muscles
and spastic paralysis.
When tetanus toxin reaches inhibitory neuron
terminals, it prevents the presynaptic release of
inhibitory neurotransmitters glycine and gamma-
aminobutyric acid (GABA).
In normal condition, Glycine and GABA released from
inhibitory interneurons induce muscle relaxation

tetanus toxin gets transported in a retrograde way (away

from axon terminals) from the peripheral nervous
system to the central nervous system

results in toxin internalization

Tetanus toxin binds to polysialo gangliosides

Tetanus notes: Drreceptors present on motor nerve terminals
Akshata G Athreya
Flaccid paralysis vs. Spastic paralysis

Flaccid paralysis occurs when the muscle

cannot contract at all. The muscle stays weak
and floppy.

Spastic paralysis occurs when the muscle stays

in contraction. The muscle is too rigid and the
patient can not move the muscle properly. It
causes muscles to twitch uncontrollably or
spasm.

Tetanus notes: Dr Akshata G Athreya

Mode of transmission
C. tetani are primarily saprophytic (live in the dead or decaying matter) and only incidentally infect
humans. There is no person-to-person spread of C. tetani.
Human acquires tetanus when Clostridium tetani endospores are inoculated into sterile body tissues through
traumas (for example, lacerations, deep puncture, or crush injuries) or fecal contamination of the
umbilical cord (neonatal tetanus). The organism would grow and produce tetanus toxin in the anoxic zones
created by local tissue death.

Tetanus notes: Dr Akshata G Athreya

 Epidemiology
Occurrence
Tetanus occurs worldwide but is most frequently encountered in densely populated regions in hot, damp climates
with soil rich in organic matter.

Reservoir
Organisms are found primarily in the soil and intestinal tracts of animals and humans.

Transmission
Transmission is primarily by contaminated wounds (apparent and inapparent). The wound may be major or
minor. In recent years, a higher proportion of tetanus cases had minor wounds, probably because severe wounds
are more likely to be appropriately managed. Tetanus may follow elective surgery, burns, deep puncture wounds,
crush wounds, otitis media, dental infection, and animal bites.

Temporal Pattern
In temperate climates, tetanus peaks in the summer. In tropical climates, tetanus generally occurs year round, but
may rise during the wet season in some areas.

Communicability
Tetanus is not contagious from person-to-person. It is the only vaccine-preventable disease that is infectious but
not contagious.
Tetanus notes: Dr Akshata G Athreya
Epidemiology
•Worldwide - present in the environment.
•In 2001 an estimated 282,000 died worldwide from tetanus, mostly in Asia, Africa and
South America.
•The incidence of tetanus in the UK decreased following the introduction of national
tetanus immunisation in 1961
•Between 1984 and 2002, there were 186 cases of tetanus in England and Wales, of which
74% occurred in individuals aged over 45 years
•Neonatal tetanus is an important cause of mortality in many countries in Asia and Africa
due to infection of the baby's umbilical stump.

Tetanus notes: Dr Akshata G Athreya

Symptoms
Complications
•Jaw cramping
•Laryngospasm (uncontrolled/involuntary tightening
•Sudden, involuntary muscle
of the vocal cords)
spasms — often in the
•Fractures (broken bones)
stomach
•Pulmonary embolism (blockage of the main artery of
•Painful muscle stiffness all
the lung or one of its branches by a blood clot that has
over the body
travelled from elsewhere in the body through the
•Trouble swallowing
bloodstream)
•Seizures (jerking or staring)
•Aspiration pneumonia (a lung infection that develops
•Headache
when things like saliva or vomit accidentally go into the
•Fever and sweating
lungs)
•Changes in blood pressure
•Breathing difficulty
and heart rate
Tetanus can lead to death (1 to 2 in 10 cases are fatal).

Tetanus notes: Dr Akshata G Athreya

Clinical and Laboratory Diagnosis

Tetanus Clinical Features

•Incubation period 8 days (range, 1 to 21 days)
•Three forms: Generalized (most common), local (uncommon), cephalic (rare)
•Generalized tetanus: Trismus (lockjaw), stiffness of the neck, difficulty swallowing, rigidity of
abdominal muscles
• Spasms continue for 3 to 4 weeks
• Complete recovery may take months

Tetanus notes: Dr Akshata G Athreya

Laboratory Diagnosis of Clostridium tetani
The clinical manifestation of tetanus and C. perfringens infection informs the laboratory diagnosis. Laboratory testing is only
performed on patients to confirm the clinical diagnosis.
Specimens
•The specimens comprise necrotic tissue fragments extracted from the depths of wounds.
•Wound swabs are not suitable specimens.
Microscopy
Gram staining of C. tetani smears is beneficial but frequently ineffective and inaccurate.
The presence of drumstick bacilli in wound tissue is not diagnostic for tetanus. This is due to:
•Some wounds may contain C. tetani without developing tetanus.
•In addition, C. tetani may not be distinguishable by microscopy from morphologically similar Clostridium species, such as C.
tetanomorphum and C. sphenoides.
Culture
•The specimens are inoculated onto blood agar and incubated for 24–48 hours under anaerobic conditions.
•C. tetani generates a swarming growth that spreads across the plate. Additionally, the specimens are inoculated into three tubes
of RCM medium, one of which is heated at 80°C for 15 minutes, the second for 5 minutes, and the third for 24–48 hours at
37°C.
•Subcultures are thereafter created everyday on blood agar for up to four days. C. tetani is isolated from the swarming margins of
blood agar colonies in pure culture.
•Because tetanus is caused by a small number of organisms and because many organisms are killed when exposed to air during
collection processing, only 30% of tetanus cases result in a positive culture.
Identification of bacteria
The distinguishing characteristics of C. tetani are their morphology, culture, and toxigenic properties.
Tetanus notes: Dr Akshata G Athreya
Toxigenicity testing
C. tetani strains are evaluated for toxin production using the following tests:

In vitro neutralization test on blood agar In vivo neutralization test in mice

Serodiagnosis
•Patients’ serum contains neither antibodies to tetanus toxin nor tetanus toxin itself, so serological tests are not performed.
Tetanus notes: Dr Akshata G Athreya
Other tests

The spatula test

•This is an extremely helpful and straightforward bedside diagnostic test for tetanus. The oropharynx is touched
with a spatula or tongue blade for this examination.
•Typically, this induces a gag reaction and the patient attempts to evacuate the spatula (negative test).
•If tetanus is present, patients suffer a reflex spasm of the masseters and bite the spatula (positive test).
•The test is 100% sensitive and 100% specific.

Identifying features of Clostridium tetani

•Extremely thin, transparent layer of growth that tends to cover the entire agar surface.
•Produce alpha-hemolytic colonies initially on blood agar, which turn beta-hemolytic after prolonged incubation
due to the synthesis of tetanolysin.
•In the Gram-stained smear of the colony, Gram-positive bacilli with conspicuous terminal spores (drumstick
look) are seen.
•Bacteria that are mobile (excluding type VI) and encapsulated.
•Avoid fermenting any sugars.
•Testing for toxicity in mice is a valid way for identifying the colony as C. tetani.

Tetanus notes: Dr Akshata G Athreya

Treatment of Clostridium tetani
It consists of antibiotic therapy and immunoglobulin therapy.
Antibiotics therapy
•Antibiotics are used to inhibit C. tetani from multiplying in the wound, hence stopping the generation and release
of toxins.
•The current antimicrobial medicine of choice is metronidazole, with penicillin serving as an option.
•Tetracycline is an alternate medication for patients with penicillin or metronidazole allergies.
•Other antimicrobials used to treat tetanus include clindamycin, erythromycin, and vancomycin.

Immunisation or Human immunoglobulin therapy

Active immunization
•Tetanus is entirely prevented by vaccination.
•Active immunisation with tetanus toxoid vaccine is the key to preventing tetanus.
•The toxoid may be administered alone or in combination with diphtheria toxoid and acellular pertussis
(whooping cough) vaccination (DTaP or triple).Tetanus notes: Dr Akshata G Athreya
Immunization for prevention of neonatal tetanus
•Tetanus toxoid is administered to previously unimmunized pregnant women twice throughout pregnancy, 4–6
weeks apart, especially in the last two trimesters and again at least 4 weeks prior to delivery.
•Antitetanus antibodies from the mother are transmitted to the foetus, and this passive immunity is effective for
several months after birth.

Passive immunization Human

•TIG (Tetanus immunoglobulin) is used to immunize passively. In nations where TIG is unavailable, ATS (Anti
toxins) is also utilized for the same purpose.

Combined immunization
•Combined immunization is achieved by administering (a) human TIG or the ATS and (b) tetanus toxoid
simultaneously.
Tetanus notes: Dr Akshata G Athreya
Vaccination is the best way to protect against tetanus
The United States sees an average of about 30 reported cases
each year. Nearly all cases of tetanus are among people who
did not get all the recommended tetanus vaccinations.
These vaccines help protect against tetanus and also provide
protection against other diseases:
•DTaP protects against diphtheria, tetanus, and pertussis
(whooping cough)
•DT protects against diphtheria and tetanus
•Tdap protects against tetanus, diphtheria, and pertussis
•Td protects against tetanus and diphtheria

CDC recommends tetanus vaccines for peopleTetanus

of all ages
notes: Dr Akshata G Athreya
Surveillance

These declines were in part because of continued use of tetanus

antitoxin for wound management and introduction of tetanus
vaccines in the 1930s and 1940s. Today, sporadic cases of
tetanus continue to occur in adults who have not gotten all
the recommended tetanus vaccinations. This includes people
who have never received a tetanus vaccine or adults who
haven’t stayed up to date on their 10-year booster shots.

Tetanus notes: Dr Akshata G Athreya

 Guide to Tetanus Prophylaxis with TIG in Routine Wound Management

Table 1
History of
adsorbed tetanus Clean, minor Clean, minor
toxoid-containing wound wound All other wounds* All other wounds*
vaccines (doses) DTaP, Tdap or Td† TIG§ DTaP, Tdap or Td† TIG§
Unknown or <3 Yes No Yes Yes
¶ **
≥3 No No No No

*Such as, but not limited to, wounds contaminated with dirt, feces, soil, or saliva; puncture wounds; avulsions; and wounds
resulting from missiles, crushing, burns, and frostbite.
†DTaP is recommended for children younger than age 7 years. Tdap is preferred to Td for persons age 11 years or older who

have not previously received Tdap. Persons age 7 years or older who are not fully immunized against pertussis, tetanus, or
diphtheria should receive one dose of Tdap (preferably the first) for wound management and as part of the catch-up series; if
additional tetanus toxoid-containing doses are required, either Td or Tdap vaccine can be used.
§People with HIV infection or severe immunodeficiency who have contaminated wounds (including minor wounds) should

also receive TIG, regardless of their history of tetanus immunizations.

¶Yes, if ≥10 years since the last tetanus toxoid-containing vaccine dose.

**Yes, if ≥5 years since the last tetanus toxoid-containing

Tetanus notes:vaccine
Dr Akshata dose.
G Athreya
In 1909 she wrote to a reporter: “I never had typhoid in my life, and
have always been healthy. Why should I be banished like a leper and
compelled to live in solitary confinement with only a dog for a
companion?”

1907 : first asymptomatic case of typhoid was identified

Dr Akshata G Athreya
Typhoid, also known as enteric fever, is a severe illness caused by salmonella bacteria.
This bacteria affects your digestive system after you consume food or water contaminated with faecal material.
The bacteria that causes typhoid fever is Salmonella bacteria. It can spread from person to person or from
consuming contaminated food. The bacteria are of two types:
1.Salmonella typhi (serotype), also known as S. typhi
2.Salmonella paratyphi serotype, which is also known as S. paratyphi serotype

Dr Akshata G Athreya
CLASSIFICATION CULTURE
Grams staining
•Domain: Bacteria
•Gram-negative pink rods (Bacillus)
•Order: Enterobacteriales
•typically move by using peritrichous flagella
•Family: Enterobacteriaceae
•non capsulated
•Order Enterobacteriales
•non spore forming, facultative anaerobe
•Genus: Salmonella
•Species: typhi, paratyphi

Colony characteristics
1.Blood agar: S.typhi and S.
paratyphi usually produce non-hemolytic
smooth white colonies on blood agar.
2.MacConkey agar: Salmonellae produce
lactose non-fermenting smooth colonies
on MacConeky agar. Dr Akshata G Athreya
Selective media are:
1. Culture may be taken from the feces.
2. MacConkey agar = pale non-lactose fermenting colonies.
3. Desoxycholate citrate agar = pale non-lactose fermenting colonies.
4. Xylose-lysine desoxycholate agar = observe for red colonies with a black center.
5. Wilson and Blair’s bismuth sulfite agar = observe black metallic colonies due to
H2S production.
6. Selenite F and tetrathionate broth for enrichment, then subculture to MacConkey
agar.

Dr Akshata G Athreya
 Biochemical Test and Identification of Salmonella typhi
Characteristics Salmonella Typhi Characteristics Salmonella Typhi
Capsule Negative (-ve) Shape Rod
Catalase Positive (+ve) Spore Negative (-ve)
Citrate Negative (-ve) TSIA (Triple Sugar Iron
Alkali/Acid
Agar)
Flagella Positive (+ve)
Urease Negative (-ve)
Gas Negative (-ve)
VP (Voges Proskauer) Negative (-ve)
Gelatin Hydrolysis Negative (-ve)
Fermentation of
Gram Staining Negative (-ve) Adonitol Negative (-ve)
Growth in KCN Negative (-ve) Arabinose Negative (-ve)
H2S Positive (+ve) Arabitol Negative (-ve)
Indole Negative (-ve) Cellobiose Negative (-ve)
Motility Motile DNase Negative (-ve)
MR (Methyl Red) Positive (+ve) Dulcitol Negative (-ve)
MUG Test Negative (-ve) Erythritol Negative (-ve)
Nitrate Reduction Positive (+ve) Esculin Hydrolysis Negative (-ve)
Oxidase Negative (-ve) Glucose Positive (+ve)
Pigment Negative (-ve) Glycerol Negative (-ve)
Dr Akshata G Athreya
Inositol Negative (-ve)
 Biochemical Test and Identification of Salmonella typhi

Characteristics Salmonella Typhi

Lactose Negative (-ve) Enzymatic Reactions
Malonate Negative (-ve) Characteristics Salmonella Typhi
Maltose Positive (+ve) Acetate Utilization Negative (-ve)
Mannitol Positive (+ve) Arginine Dehydrolase Negative (-ve)
Mannose Positive (+ve) Esculin Hydrolysis Negative (-ve)
Melibiose Positive (+ve) Lipase Negative (-ve)
Mucate Negative (-ve) Lysine Positive (+ve)
MyoInositol Negative (-ve) ONPG (β-galactosidase) Negative (-ve)
Raffinose Negative (-ve) Ornithine Decarboxylase Negative (-ve)
Rhamnose Negative (-ve) Peroxidase Negative (-ve)
Salicin Negative (-ve) Tyrosine Hydrolysis Negative (-ve)
Sorbitol Positive (+ve)
Sucrose Negative (-ve)
Tartrate Positive (+ve)
Trehalose Positive (+ve)
Xylose Positive (+ve)
Dr Akshata G Athreya
 Results of Salmonella spp in Triple Sugar Iron (TSI) Agar

Glucose not
fermented,formation of H2S
happens
Results of Salmonella spp in Motility Indole Urease (MIU) medium and citrate utilization test

Organism Motility Indole Urease Citrate utilization test

S.typhi +ve -ve -ve -ve
S.paratyphi A +ve -ve -ve +ve
Other Salmonella spp +ve -ve
Dr Akshata G Athreya -ve variable
 EPIDEMIOLOGY

Dr Akshata G Athreya
AN OVERVIEW:

WHO estimates the global typhoid fever disease burden at 11-20 million cases annually, resulting in

about 128,000–161,000 deaths annually. In recent years, data indicates that typhoid fever is a major

cause of morbidity among the urban and peri-urban populations and the single most important cause of

febrile bacteraemia fever caused by bacteria in the bloodstream.

Dr Akshata G Athreya
Symptoms typically begin 1–3 weeks after exposure to the bacteria.
The two main symptoms of typhoid are fever and rash. Typhoid fever is particularly high, gradually
increasing over several days up to 104ºF.
The rash, which does not affect every person, consists of rose-colored spots, particularly on the neck and
abdomen.
Other symptoms can include:
•diarrhea
•loss of appetite
•bloating
•nausea
•weakness
•abdominal pain
•constipation
•headaches

Possible Complications
Health problems that may develop include:
•Intestinal hemorrhage (severe GI bleeding)
•Intestinal perforation
•Kidney failure
•Peritonitis Dr Akshata G Athreya
Causes
✓ Typhoid is caused by the bacteria S. typhi. It spreads through food, drinks, and drinking water
that are contaminated with infected fecal matter.
✓ Washing fruit and vegetables can spread it as well if the water is contaminated.
✓ Some people have typhoid without experiencing any symptoms. Others continue to harbor the bacteria
after their symptoms have gone. Sometimes, the disease can appear again.
✓ People who test positive for typhoid may not be allowed to work with children or older adults until
medical tests are negative.

Dr Akshata G Athreya
 Antigenic components

S. Typhi has two antigens named:

1.S. typhi O (TO), primary antigen
2.S. typhi H (TH), secondary antigen
On the other hand, the bacteria S. Paratyphi has
the following two antigens:
1.S. Paratyphi A (AH)
2.S. Paratyphi B (BH)

Dr Akshata G Athreya
 ANTIGENIC COMPONENTS
Salmonella serotypes, the bacteria has been shown to possess three types of antigen.
H (flagella antigen), antigen O (somatic antigen) and Vi (capsular). These antigens play an important role when
it comes to grouping or serotyping the organisms
•O antigen: This antigen is composed of lipopolysaccharide. Also refered to as somatic antigen, O antigen occurs
on the outer membrane and is typically determined by the sugar sequence. Less immunogenic and cross
reactive

•H antigen - Known as flagellar antigen. This includes the proteins that are found on the flagella of the bacteria.
The H antigen occurs as either phase 1 or phase 2 (or both in some cases). Strongly antigenic, they rapidly rise
the antibodies when infected, last long and are specific. Several serovars are present.
•Vi antigen - Virulence antigen. Vi is found in a few serovars and is a superficial antigen that overlies the O
antigen. As such, it is an additional antigen found in such organisms as Salmonella typhi and Salmonella
paratyphi C where it plays an important role in confirming serotype (serotype : differ with the surface antigens)
determination. Poorly immunogenic, disappears , very rarely
Dr Akshata seen in chronic cases
G Athreya
 Pathology of salmonella (Enteric fever):

Dr Akshata G Athreya
PATHOGENECITY
Pathogenesis of typhoid fever:
1.There are three types of disease:
1. Enteric fever.
2. Gastroenteritis.
3. Septicemia.
2.This organism crosses the intestinal epithelium (invades the regional lymph nodes)
multiply in the macrophagic cells of Peyer’s patches, mesenteric lymph nodes, and
spleen.
3.The Peyer’s patches are inflamed and may ulcerate.
4.Bacteremia occurs, and the infection spreads to the lungs, gallbladder, kidneys,
and CNS.
5. During this invasion, these bacteria are phagocytosed by the monocytes (mononuclear
phagocytes) and can survive intracellularly, so these are called facultative intracellular
parasites.
6.Enteric fever is caused by S.typhi and S. paratyphi
7.These bacteria cause gastroenteritis, food poisoning, and a few special species that may
cause septicemia. Dr Akshata G Athreya
Dr Akshata G Athreya
Clinical diagnosis of 1st week illness Clinical diagnosis of 2nd week illness

Dr Akshata G Athreya
Clinical diagnosis of 3rd week illness

Dr Akshata G Athreya
 Lab diagnosis
A complete blood count (CBC) will show a high number of white blood cells.
A blood culture during the first week of the fever can show S typhi bacteria.
Other tests that can help diagnose this condition include:
•ELISA blood test to look for antibodies to the S typhi bacteria
•Fluorescent antibody study to look for substances that are specific to S typhi bacteria
•Platelet count (platelet count may be low)
•Stool culture

Dr Akshata G Athreya
LABORATORY DIAGNOSIS

CULTURE
• Blood culture is the mainstay for the diagnosis of Typhoid
fever.
• Blood for culture should be taken before the patient is given
antimicrobial therapy.
• Patients with a history of fever for 7 to 10 days are more
likely than others to have a positive blood culture.
• Definitive diagnosis of typhoid fever depends on the
isolation of S.typhi from blood or bone marrow aspirate
culture.
Dr Akshata G Athreya
 Widal Test- Introduction, Principle, Procedure, Interpretation and Limitation

The main principle of widal test is that if homologous antibody is present in patients serum, it will react with
respective antigen in the reagent and gives visible clumping on the test card and agglutination in the tube.
The antigens used in the test are “H” and “O” antigens of Salmonella Typhi and “H” antigen
of S. Paratyphi. The paratyphoid “O” antigen are not employed as they cross react with typhoid “O”
antigen due to the sharing of factor 12. “O” antigen is a somatic antigen and “H” antigen is flagellar antigen.

•The titre of the patient serum using Widal test antigen suspensions is the highest dilution of the serum sample
that gives a visible agglutination.
•The sample which shows the titre of 1:100 or more for O agglutinations and 1:200 or more for H
agglutination should be considered as clinically significant (active infection). Example: In figure, titre is 160.
•H agglutination is more reliable than O agglutinin.
•Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre
remains steady till 4th week after which it declines.
Dr Akshata G Athreya
Dr Akshata G Athreya
Dr Akshata G Athreya
Dr Akshata G Athreya
 Treatment
The only effective treatment for typhoid is antibiotics. Doctors most commonly use ciprofloxacin (Cipro) for
nonpregnant people.
Other antibiotics a doctor may use are:
•chloramphenicol (Chloromycetin)
•ampicillin (Ampi, Omnipen, Penglobe, and Principen)
•sulfamethoxazole/trimethoprim (Bactrim)
Pregnant people should also avoid chloramphenicol.
A person with typhoid needs to rehydrate by drinking adequate amounts of water. In more severe cases, where
the bowel has become perforated, a person may need surgery.
However, as with a number of other bacterial conditions, there is concern about the growing resistance of
antibiotics to S. typhi.
Dr Akshata G Athreya
Vaccination
Before traveling to a high risk area, a person should receive a vaccine against typhoid fever.

The typhoid vaccine is available as an oral medication or a one-off injection:

•Capsule: For adults and children over the age of 6 years, this is a live, attenuated vaccine. It consists of four
tablets that a person should take every other day, the last of which at least 1 week before travel. However,
the capsule version is currently not available in the U.S.
•Shot: over the age of 2 years, this is an inactivated vaccine a person needs to get 2 weeks before travel. A
person who has previously received the vaccine should get a booster shot 2 weeks before traveling.
The typhoid vaccine is only 50–80% effective, so a person should still exercise caution when eating, drinking,
and coming into contact with people.
Anyone living with HIV should not take the live, oral dose. The vaccine may also have adverse effects.

Dr Akshata G Athreya
Dr Akshata G Athreya
 Prophylaxis
What are three ways to prevent typhoid?
Getting vaccinated
Choosing food and drinks carefully
Washing your hand

Dr Akshata G Athreya
Dr Akshata G Athreya