Widal test is most common test to diagnose typhoid fever.

2. WIDAL TEST
Talha Raza
Bsc (HONS) MLT
FMH institute of allied health sciences
Email: talharaza.mlt@gmail.com

3. INTRODUCTION OF WIDAL TEST
 Widal test is a serological test that is done for the diagnosis of
typhoid fever caused by Salmonella organism.
 This test detects the “O” and “H” antigen of Salmonella typhi and
paratyphi A,B and C.
 When facilities for culturing are not available, the Widal test is the
reliable and can be of value in the diagnosis of typhoid fevers in
endemic areas.

4. CLINICAL SIGNIFICANCE
• Typhoid fever or enteric fever occurs when S. Typhi, S. Paratyphi
A, S. Paratyphi B, S. Paratyphi C infect the human body.
• Body responds to this antigenic stuimulus by producing
antibodies whose titre rise slowly in early stages, to a maximum
and then slowly falls till it is undetectable.
• 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

5. TYPHOID FEVER
History:
• Thomas Willis who is credited with the
first description of typhoid fever in 1659.
• French physician Pierre Charles
Alexandre Louis first proposed
the name “typhoid fever”

6. CONT.
The best known carrier was "Typhoid
Mary“; Mary Mallon was a cook in Oyster
Bay, New York in 1906 who is known to
have infected 53 people, 5 of whom died.
2. Later returned with false name but
detained and quarantined after another
typhoid outbreak.
3. She died of pneumonia after 26 years in
quarantine.

9. HOW DOES BACTERIA CAUSE
Ingestion of contaminated food or water
Salmonella bacteria
Invade small intestine and enter the bloodstream
Carried by white blood cells in the liver, spleen, and bone
marrow
Multiply and reenter the bloodstream

10. CONT.
Bacteria invade the gallbladder, biliary system, and the lymphatic
tissue of the bowel and multiply in high numbers
Then pass into the intestinal tract and can be identified for
diagnosis in cultures from the stool tested in the laboratory

12. DIAGNOSIS
Diagnosis of typhoid fever is made by
• Blood culture
• Stool culture
• Bone marrow culture
• Widal test
• Typhidot (ICT/ELISA)
• Antimicrobial susceptibility testing

13. HISTORY
 It was developed by Georges Ferdinand Widal
in 1896.

14. PRINCIPLE
• “When the colored, smooth, killed Salmonella antigen
suspensions are mixed/incubated with patient serum, anti
salmonella antibody present in the patient serum react with the
antigen suspensions to give agglutination. Agglutination is a
positive test result, indicating presence of anti salmonella
antibodies in the patient serum. No agglutination is a negative
test result indicating absence of anti salmonella antibodies”.

15. • The patient’s serum is tested for O and H antibodies (agglutinins)
against the following antigen suspensions (stained suspensions):
•
S. Typhi 0 antigen
S. Typhi H antigen
• S. Paratyphi A H antigen
S. Paratyphi B H antigen
S. Paratyphi C H antigen
• The paratyphoid “O” antigen are not employed as they cross react with
typhoid “O” antigen.
• “H” suspensions are colored red while “O” suspensions color is “Blue”.

16.  Specimen Collection and Precautions
• No special preparation of the patient is required prior to sample
collection.
• Don’t use hemolysed sample.
• Don’t heat or inactivate the serum.
• Freshly collected sample is a preferable, store at 2-8 C for up to 72
hours in case of delay in testing.
• It is preferable to test two specimens of sera at an interval of 7 to 10
days to demonstrate a rising antibody titer.
• Salmonella antibody starts appearing in serum at the end of first week
and rise sharply during the 3rd week of endemic fever.
• In acute typhoid fever, O agglutinins can usually be detected 6–8 days
after the onset of fever and H agglutinins after 10–12 days.

17. REQUIREMENTS
 Reagents
commercially prepared antigen suspension of
• S. typhi O (2-8°C)
• S. typhi H (2-8°C)
• S. paratyphi AH (2-8°C)
• S. paratyphi BH (2-8°C)
• Positive control (2-8°C)
• Negative control (2-8°C)
 Glass slide/test card
 Disposable mixing sticks
 Test tubes

18. STORAGE:
• Store all reagents at 2-8◦C.
• Read carefully expiry date mentioned on kit before use.
• Bring all reagents at room temperature, gently shake and mix before
use.
NOTE:
• The S. Typhi “O” reagent contains 0.5 % phenol, S. Typhi “H”, S.
paratyphi “AH”, “BH” reagents contains 0.3 % formaldehyde as
preservative.
• Avoid contact with skin and mucosa.

19. COMMERCIALLY PREPARED WIDAL TEST KIT

20. PROCEDURE
1) Slide method
2) Tube method

21. SLIDE METHOD
 QUALITATIVE METHOD
 Place one drop of positive control on one reaction circle of test card or on
slide.
 Pipette one drop of Normal saline on next circle for negative control.
 Pipette one drop of patient serum onto remaining four reaction circles.
 Add one drop of H suspension on 1st and 2nd reaction circle (on +ve and –
ve control circle)
 Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four
reaction circles.
 Mix contents of each circle uniformly over the entire circle with separate
mixing sticks.
 Rotate the slide, gently back and forth and observe for agglutination
macroscopically within one minute.

22. AGGLUTINATION REACTION FOR DIFFERENT ANTIGEN SOLUTIONS WITH TEST SAMPLE

23.  SEMI QUANTITATIVE METHOD
 Pipette 5, 10, 20, 40, 80 ul of the test sample on the reaction circles.
 Add to each reaction circle, a drop of the antigen which showed
agglutination with the test sample in the screening method.
 Using separate mixing sticks, mix the contents of each circle uniformly
over the reaction circles.
 Rock the slide gently back and forth, observe for agglutination
macroscopically within one minute.

24. Serum volume Titre
80 ul 1:20
40 ul 1:40
20 ul 1:80
10 ul 1:160
05 ul 1:320

25. STANDARD TUBE METHOD
Take four sets of 8 test tubes and label them 1 to 8 for O,H,AH and BH antibody
detection.
Pipette in to the tube No.1 of all sets 1.9 ml of isotonic saline.
To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
To the tube No. 1 tube in each row add 0.1 ml of the serum sample to be tested and mix
well.
Transfer 1ml of the diluted serum from tube no.1 to tube no.2 and mix well.
Discard the 1ml of the diluted serum from tube no.7 of each set.

26. Tube no.8 in all sets serves as a saline control. The dilution
of the serum sample achieved in each set is as follows:
Tube no. 1 2 3 4 5 6 7 8 (control)
Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280
To all tubes (1 to 8) of each set add one drop of the respective WIDAL TEST antigen
suspension (O,H,AH,BH) from reagent vials and mix well.
Cover the tubes and incubate at 37 C overnight (approx. 18 hrs).
Dislodge the sedimented button gently and observe.

28. INTERPRETATION
• Agglutination indicates positive test result.
• Significant titre is >1:160 but varies from population to population.
• Titre of “O” antigen more than 1:160 indicates recent infection.
• Titre of “H” antigen more than 1:160 indicates past infection or due to
immunization.
• False positive result may be in vaccination or previously infection with S.
typhi.
• False negative may be due to antimicrobial treatment which blocks
antibody response.