Faculty of Allied Medical

Sciences
Clinical Immunology & Serology
Practice
(MLIS 201)

Typhoid fever

Prof. Dr. Ezzat M Hassan

Prof. of Immunology
Med Res Inst, Alex Univ
E-mail: elgreatlyem@hotmail.com

Teaching Objectives

To define Typhoid Fever

To know the causes and symptoms of the disease
To understand how the bacteria cause the disease
To know different methods to diagnose Typhoid fever
To define Widal test and its components
To describe the procedure of tube Widal test
To know how to interpret the results
To describe the procedure of slide Widal test
To know the causes of false +ve and flase ve results
To know the limitations of the test
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Countries endemic for typhoid(U.S. CDC 2006)

Alex LaPointe, Wikimedia Commons

Definition
An infectious feverish disease caused by the bacterium Salmonella typhi
and less commonly by Salmonella paratyphi.
The infection always comes from another human, either an ill person or
a healthy carrier of the bacterium.

Persons with typhoid fever carry the bacteria in their bloodstream and
intestinal tract

Transmitted through the ingestion of food or drink contaminated by the

feces or urine of infected people
The bacterium can withstand both drying and refrigeration.

Causes
1. Caused by the bacterium Salmonella Typhi .
2. Ingestion of contaminated food or water.
3. Contact with an acute case of typhoid fever.
4. Contact with a chronic asymptomatic carrier.

Salmonella typhi

Rod shaped, flagellated, aerobic, Gram -ve bacilli.

Refrigeration and freezing could slow their growth.
Pasteurizing and food irradiation kill Salmonella for
commercially-produced foodstuffs
Foods prepared in the home from raw eggs can spread
salmonella if not properly cooked before consumption.

How does the bacteria cause disease ?

Ingestion of contaminated food or water
Salmonella typhi
Carried by white blood cells into the liver, spleen, and bone marrow
Multiply and reenter the bloodstream (Clinical illness)
Bacteria invade the gallbladder, biliary system, and the lymphatic tissue of the
bowel and multiply in high numbers
Then pass into the intestinal tract (can be identified for diagnosis in cultures
from the stool)
Typhoid ulcers can cause perforation and hemorrhage

How does the bacteria cause disease ?

Symptoms
No symptoms - if only a mild exposure; some people
become "carriers" of typhoid.
Poor appetite, Headaches and generalized pains,
Fever, Lethargy
Rose spots on chest wall
Diarrhea / constipation and abdominal pain
Chest congestion develops in many patients,
slow heartbeat.
Enlarged spleen and liver

Symptoms

Aches and pains

Rose spots

High fever

Diarrhea

Chest congestion

Typhoid Meningitis

Diagnosis
Diagnosis of typhoid fever is made by

Clinical examination
Blood, bone marrow, or stool cultures for S. typhi
Serological Tests

Serodiagnosis of Typhoid :
1.Detection of Antibodies in serum:
1.Widal test (Tube or Slide),
3.Tubex system,

2.Typhidot assay
4. Dipstick assay.

2. Detection of Antigens in serum:

1. Tubex system
2. Countercurrent Immunoelectrophoresis (CIE).
3. Co-agglutination test.
4. ELISA

3. Detection of Antigens in urine:

1.Tubex system
3. Latex agglutination

2. CIE,
4. Co-agglutination

Widal test

 Antigenic structure of Salmonella

H( flagella )
antigens
O (somatic)
antigens
Vi (Virulence)
capsular
polysaccharide
antigens

O (somatic) antigens

LPS in the cell wall;

Heat stable
Less immunogenic

Agglutination with
antisera:
Fine, compact, granular
chalky clumps

H (flagella) antigens

Present in flagella;
Heat labile;
Strongly immunogenic;
Induce rapid & High Ab
titres;

Agglutination with
antisera:
Large, loose, cotton
wool clumps

Vi (virulence) antigen
Capsular polysaccharide expressed on certain serotypes
Heat labile;
Poorly immunogenic, BUT antibodies are protective:
1. Detection of Vi antibody not helpful in diagnosis
2. Absence in a case of typhoid poor prognosis;
3. Persistence of Vi antibody : carrier state

WIDAL Test

Tube agglutination test.

Detects anti O and H antibodies in serum
Diagnosis of Typhoid and Paratyphoid cases
Carriers of typhoid bacilli possess antibody against
the Vi antigen of S. typhi. (Vi tires seem to correlate
better with the carrier state than do O or H titres).
For this reason, the use of Vi agglutination for
detection of carriers was suggested .

Dr.T.V.Rao MD

19

Widal test
Significance
I st week negative.
Titers raise in 2nd week
Raise of titers is diagnostic

Dr.T.V.Rao MD

20

Materials
Antigens:

Suspension of S. typhi "O" antigen, O

Suspension of S. typhi "H" antigen; H
Suspension of S. paratyphi A "H" antigen, PA
Suspension of S. schottmuelleri "H" antigen, PB

Antibody: serum of suspected patient

Normal saline
Test tubes and pipettes

PROCEDURE
1
O
H
PA
PB

PROCEDURE
Make the mark of tubes
Dilute patients serum 1:10 (0.1 ml serum + 0.9 ml saline).
Add reagents as the following:
Reagent (ml)
1
2
3
4
5
6
7
Saline
0.5
1:10 Patient serum 0.5
Serum dilution
1:20
Bacteria suspension
0. 5

0. 5
0.5
0. 5
0. 5 0. 5
0.5
0.5
0.5
0.5
0. 5
1:40 1:80 1:160 1:320 1:640
0. 5 0. 5 0. 5
0. 5 0. 5

Final serum dil.

1:80 1:160 1:320 1:640 1:1280

1:40

0. 5
discard

0. 5
-ve

Bacteria suspension: O H PA PB
Shake several times, put it in 370C water bath for 16-18 hours.
Then let it stand at room temperature over night .

Observation:
**Do not shake tubes before reading the results
1. Control tube (Tube No. 7): no agglutination (-)
2. Lowest titer tube: absolutely agglutination (++++)
3. Other tubes:
agglutination(+++) agglutination (++)
agglutination (+)
no agglutination (-)

Interpretation:
Agglutination titer: the highest dilution of serum
which appears (++) bacteria agglutination.

Agglutination how it appears

after reactivity
O

Felix tube
Round bottom
O agglutination

Compact
granular
agglutination

Dreyers tube
Conical bottom
H agglutination

Loose
Cotton woolly
clumps

Observed for agglutination:

H : Loose , cotton woolly clumps;
O : Compact Fine granular
agglutination;

How do you read Widal test results for

typhoid fever?
The highest dilution of the patients serum in
which agglutinations occurs is noted, ex. if the
dilution is 1 in 160 then the titer is 160.
Agglutination in dilution up to <1:60 is seen
in normal individuals . Agglutination in dilution
1:160 is suggestive of Salmonella infection.
Agglutination in dilution of >1:320 is
confirmatory of Enteric fever .

Interpretation of results
O<1:80, H<1:160,

Normal value

PH<1:80
O 1:80 & H 1:160 or

Typhoid fever

O 1:80 & PH 1:80

Paratyphoid fever

O 1:80 & H <1:160 or

Early infection or other

O 1:80 & PH <1:80

salmonella infections

O <1:80 & H 1:160 or

Vaccination or nonspecific

O <1:80 & PH 1:80

memory reaction

Conclusion
1

++++

++

++

++++

+++

++

++

PA

++

PB

1:40

1:80

1:160 1:320 1:640 :1280 Negative

control

N.B.
Single test not diagnostic (Except at high titers > 320).
Paired samples tests with rising titer is diagnostic
Diagnostic.
O > 1 : 80
H > 1 :160
H agglutinins appear first

Prozone phenomenon in
Agglutination tests
Prozone effect - Occasionally, it is observed that when
the concentration of antibody is high (i.e. lower
dilutions), there is no agglutination and then, as the
sample is diluted, agglutination occurs.
Lack of agglutination in the prozone is due to antibody
excess resulting in very small complexes that do not
clump to form visible agglutination

Limitation of Widal Test

The Widal test is time consuming and often times
when diagnosis is reached it is too late to start an
antibiotic regimen.
In spite of several limitation many Physicians
depend on Widal Test

Dr.T.V.Rao MD

31

Interpretation of Widal test

Test results need to be interpreted
carefully in the light of :
1. Past history of enteric fever,
2. Typhoid vaccination,
3. general level of antibodies in the
healthy populations in endemic areas
of the world.

False Positive Reactions with

WIDAL Test
1. patients who have had previous vaccination or
infection with S typhi.
2. Cross-reaction with non typhoidal Salmonella.
3. in association with some autoimmune diseases.
4. Infection with malaria

False Negative Reactions with WIDAL

Test
1. Early treatment,
2. Relapses of typhoid fever.
3. Occasionally the infecting strains are
poorly immunogenic.

Typical Serological Profile After Acute

Infection

Note that during Reinfections, IgM may be absent or present at a low level transiently

Slide Widal test:

Slide Widal test is more popular as it gives rapid results.
Qualitative test:
1 drop of undiluted patients serum sample for
the 2 antigens is placed on the circled card.
1 drop of each of 2 salmonella antigens are added separately
rotated gently for 1 min.
Appearance of agglutination gives qualitative results.
(semiquantitative test is repeated with dilutions of serum)

Slide Widal test (Cont.):

Semi-quantitative test:
80l, 40l, 20l, 10l, 5l, of patients serum each for 2 salmonella antigens are placed on the
circled card.

one drop of specific antigen is added to each series of serum.

Agglutination of each of these is noted.

Interpretation
80l corresponds to 1 in 20 dilution.
40l corresponds to 1 in 40 dilution.
20l corresponds to 1 in 80 dilution.
10l corresponds to 1 in 160 dilution.
5l corresponds to 1 in 320 dilution.

Prevention
And
Treatment

Prevention

Two main typhoid fever prevention strategies:

1. Vaccination
.
2. Avoid risky food and drinks

Medication
Antibiotics

Antibiotics, such as ampicillin, chloramphenicol, fluoroquinolone

trimethoprim-sulfamethoxazole, Amoxicillin and ciprofloxacin etc used to
treat typhoid fever.

Prompt treatment of the disease with antibiotics reduces the casefatality rate to approximately 1%.

Study Questions:
Write short note:
Symptoms & Prevention of typhoid fever

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Assigment
Write shortly on Widal test

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THANK YOU

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