2. Faculty of Allied Medical Sciences
Clinical Immunology & Serology Practice
(MLIS 201)

3. Typhoid fever Prof. Dr. Ezzat M Hassan Prof. of Immunology
Med Res Inst, Alex Univ

4. 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

5. Countries endemic for typhoid (U.S. CDC 2006)
Alex LaPointe, Wikimedia Commons

6. 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.

7. 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.

8. 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.

9. 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

10. How does the bacteria cause disease ?

11. 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

12. Symptoms Aches and pains Rose spots High fever Diarrhea
Chest congestion
Typhoid Meningitis

13. Diagnosis Clinical examination
Diagnosis of typhoid fever is made by
Clinical examination
Blood, bone marrow, or stool cultures for S. typhi
Serological Tests

14. Serodiagnosis of Typhoid :
1.Detection of Antibodies in serum:
1.Widal test (Tube or Slide), Typhidot assay
3.Tubex system, Dipstick assay.
2. Detection of Antigens in serum:
1. Tubex system Countercurrent Immunoelectrophoresis (CIE).
3. Co-agglutination test ELISA
3. Detection of Antigens in urine:
1.Tubex system CIE,
3. Latex agglutination Co-agglutination

15. Widal test

16. Antigenic structure of Salmonella
H( flagella ) antigens
O (somatic) antigens
Vi (Virulence) capsular polysaccharide antigens

17. O (somatic) antigens H (flagella) antigens
LPS in the cell wall;
Heat stable
Less immunogenic
Agglutination with antisera:
Fine, compact, granular chalky clumps
Present in flagella;
Heat labile;
Strongly immunogenic;
Induce rapid & High Ab titres;
Agglutination with antisera:
Large, loose, cotton wool clumps

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

19. 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

20. Widal test Significance I st week negative. Titers raise in 2nd week
Raise of titers is diagnostic
Dr.T.V.Rao MD

21. Materials Antigens: Antibody: serum of suspected patient Normal saline
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

22. PROCEDURE 1 2 3 4 5 6 7 O H PA PB

23. PROCEDURE Make the mark of tubes
Dilute patient’s serum 1:10 (0.1 ml serum ml saline).
Add reagents as the following:
Reagent (ml)
Saline
1:10 Patient serum discard
Serum dilution :20 1:40 1: :160 1: :
Bacteria suspension
Final serum dil : :80 1:160 1: :640 1: ve
Bacteria suspension: O H PA PB
Shake several times, put it in 370C water bath for hours. Then let it stand at room temperature over night.

24. Observation: Interpretation:
**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:
¾ agglutination(+++) ½ agglutination (++)
¼ agglutination (+) no agglutination (-)
Interpretation:
Agglutination titer: the highest dilution of serum
which appears (++) bacteria agglutination.

25. Agglutination how it appears after reactivity
Dreyer’s tube
Conical bottom
H agglutination
Felix tube
Round bottom
O agglutination
Compact
granular
agglutination
Loose
Cotton woolly
clumps
Observed for agglutination:
H : Loose , cotton woolly clumps;
O : Compact Fine granular agglutination;
Supernatant should be clear;

27. 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 .

28. Interpretation of results
O<1:80, H<1:160, PH<1:80
Normal value
O ≥1:80 & H ≥1:160 or
O ≥1:80 & PH ≥1:80
Typhoid fever
Paratyphoid fever
O ≥1:80 & H <1:160 or
O ≥1:80 & PH <1:80
Early infection or other salmonella infections
O <1:80 & H ≥1:160 or
O <1:80 & PH ≥ 1:80
Vaccination or nonspecific memory reaction

29. Conclusion 1 2 3 4 5 6 7 O ++++ ++ ++ + - - - H ++++ +++ ++ ++ - - - O H PA PB
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

30. 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

31. 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

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

33. False Positive Reactions with WIDAL Test
patients who have had previous vaccination or infection with S typhi.
Cross-reaction with non – typhoidal Salmonella.
in association with some autoimmune diseases.
Infection with malaria

34. False Negative Reactions with WIDAL Test
Early treatment,
Relapses of typhoid fever.
Occasionally the infecting strains are poorly immunogenic.

35. Typical Serological Profile After Acute Infection
Note that during Reinfections, IgM may be absent or present at a low level transiently

36. Slide Widal test:
Slide Widal test is more popular as it gives rapid results.
Qualitative test:
1 drop of undiluted patient’s 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)

37. Slide Widal test (Cont.):
Semi-quantitative test:
80µl, 40µl, 20µl, 10µl, 5µl, of patient’s 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
80µl corresponds to 1 in 20 dilution.
40µl corresponds to 1 in 40 dilution.
20µl corresponds to 1 in 80 dilution.
10µl corresponds to 1 in 160 dilution.
5µl corresponds to 1 in 320 dilution.

38. Prevention
And
Treatment

39. Prevention Two main typhoid fever prevention strategies:
1. Vaccination . 
2. Avoid risky food and drinks

40. 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 case-fatality rate to approximately 1%.

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

42. Assigment Write shortly on Widal test
ميرنا ابراهيم – نادية محمد سعد – نيهال كمال – هايدى احمد – هدى عبد الله

43. THANK YOU