The Widal test detects antibodies in patient serum that agglutinate Salmonella typhi and paratyphi antigens. It was developed in 1896 by Georges Widal and is useful for diagnosing typhoid fever in endemic areas without culture facilities. The test involves mixing patient serum with O and H antigens from S. typhi and S. paratyphi and observing for agglutination. A rising antibody titer on repeat testing supports a diagnosis of typhoid fever.

1. WIDAL TEST
PRESENTED BY: ASMITA PRAJAPATI
3RD YEAR BSC. MLT
JFIHS/ LACHS

2. INTRODUCTION
 Widal test is a common agglutination test which is
employed in the diagnosis of enteric fever caused by
Salmonella typhi & Salmonella paratyphi A, B & C.
 It detects the presence of serum agglutinins i.e. ‘O’ &
‘H’ in patients serum with typhoid & paratyphoid fever.

3.  This test was developed by Georges Ferdinand Widal in 1896.
 It is reliable & can be
of value in the diagnosis
of typhoid fevers in
endemic areas when
facilities for culturing are
not available.

4.  Salmonella antibody starts appearing in the serum at the end of 1st
week & 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 & H agglutinins after 10-12 days.
 It is preferable to test two specimens of sera at an interval of 7-10
days to demonstrate a rising antibody titre.

5. PRINCIPLE
 Its main principle is that if homologous Ab is present in patients
serum, it will react with respective Ag in the reagent & gives visible
clumping on the test card & agglutination in the tube.
 The Ags used in the test are ‘H’ & ‘O’ Ags of S. typhi & ‘H’ Ag of S.
paratyphi. ‘O’ is a somatic Ag & ‘H’ is a flagellar Ag.
 The ‘O’ Ag of S. paratyphi is not used as it cross reacts with the ‘O’
Ag of S. typhi due to the sharing of factor 12.

6. PREPARATION OF WIDAL
ANTIGENS
 The Ag suspensions used for the Widal test may be
prepared from suitable stock cultures in the lab. But
generally commercially prepared Ags are used.
 S. typhi is used to prepare S. typhi ‘O’ & ‘H’ Ags.
 H suspension of bacteria is prepared by treating
overnight broth culture or saline suspension of
Salmonella with 0.1% formalin.

7.  For preparation of ‘O’ suspension of bacteria, the organism is
cultured on phenol agar (1:800) to inhibit flagella. The growth is
then emulsified in small volume of saline, mixed with 20X its volume
of alcohol, heated at 40-50°C for 30 mins. & centrifuged.
 The Ags thus prepared are treated with chloroform (preservative) &
appropriate dyes are added for easy identification of Ags.

8. METHODS OF WIDAL TEST
 It can be performed in 2 different ways:
1. Slide agglutination Widal test
a) Qualitative slide test
b) Quantitative slide test
2. Tube agglutination Widal test
• Tube agglutination test has more accuracy as compared to the
slide agglutination technique.
• However, a slide Widal test is more popular among diagnostic
laboratories as it gives rapid results.

9. Slide Agglutination Test
1. Qualitative slide agglutination test
Procedure:
 Firstly, bring all reagents to room temperature & mix well.
 Add 1 drop of test sample (25µL) into each reaction circle labeled as O, H,
AH &BH according to given Ag solution.
 Add 1 drop of PC into the circle marked as PC & 1 drop of isotonic saline on
the next reaction circle marked as NC.
 Add 1 drop of ‘H’ suspension to PC & NC and 1 drop of ‘O’, ‘H’, ‘AH’ & ‘BH’
Ag to the respective circles labeled.
 Mix thoroughly with the aid of applicator stick.
 Observe for agglutination.

10. Interpretation
 Positive test: Agglutination within a minute.
 Negative test: No agglutination.

11. 2. Quantitative slide agglutination test
Procedure:
 Bring all reagents to room temperature to RT & mix well.
 Dispense 1 drop of isotonic saline to the 1st reaction circle & then place 5, 10,
20, 40, 80µL of the test sample on the remaining circles.
 Now, add 1 drop of the Ag, which showed agglutination with the test sample
in the qualitative (screening) method, to each circle.
 Mix the contents of each circle with the aid of applicator stick & rotate
gently.
 Observe for agglutination.

12. Interpretation
 The Ab titre of the test sample is its highest dilution that gives a visible
agglutination.
 80µL corresponds to 1 in 20 dilution, 40µL to 1 in 40, 20µL to 1 in 80, 10µL to
1 in 160 & 5µL corresponds to 1 in 320 titre.
 Agglutinin titre >1:80 is considered as significant infection & low titre
indicate absence of infection.

13. Quantitative tube agglutination Widal
test
 In Widal test, 2 types of tubes were originally used:
1. Dreyer’s tube (narrow tube with conical bottom) for H agglutination &
2. Felix tube (short round-bottomed tube) for O agglutination.
 Nowadays, 3 x 0.5 mL Kahn tubes are used for both types of agglutination.

14. Procedure for Quantitative tube test:
 Firstly, bring all the reagents to RT.
 Prepare 4 sets of 8 Kahn tubes/test tubes & label them 1 to 8 for O, H,
AH & BH Ab detection.
 Pipette 1.9mL of isotonic saline to tube no. 1 of each antigen set.
 Then, to tube no. 2-8 of all sets add 1mL of isotonic saline.
 To the tube 1 of each set, add 0.1mL of the serum sample to be tested &
mix well.
 Then, transfer 1.0mL of the diluted serum from tube 1 to tube 2 & mix
well.

15.  Then, transfer 1.0mL of the diluted serum from tube 2 to tube 3 & mix
well. Continue this serial dilution till tube 7 in each set.
 Discard 1.0mL of the diluted serum from tube no. 7 in each set.
 Tube no. 8 in all the sets, serves as a saline control.
 Now, the dilution of the serum sample achieved in each set is as follows:
Tube no. 1 2 3 4 5 6 7 8(Contro
)
Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280

16.  To all the tubes (1 to 8) of each set add 1 drop of the respective Widal
test Ag suspension (O, H, AH & BH) from the reagent vials & mix well.

17.  Cover the tubes & incubate overnight at 37°C (approx. 18 hrs.).
 Dislodge the sediment after incubation & observe for agglutination.
 Interpretation
 Agglutinin titre greater than 1:80 is considered as significant infection &
low titre indicate the absence of infection.

18. LIMITATIONS OF WIDAL TEST
 It is done within 7 days of illness & after 4 weeks are usually negative.
 The local titre of the place should be known for the results to be
interpreted correctly.
 The quantitative tube method of this test is highly time consuming.
 Previous typhoid vaccine may contribute to elevated agglutinins in the
non-infected population.
 Other infections of non-enteric Salmonella infections such as typhus,
immunological disorders, chronic liver disease may cause false positive
result. Cross reaction between malaria parasites & Salmonella Ags may
also cause false positive agglutination test.