WIDAL TUBE TEST

 

WIDAL TUBE TEST

Introduction

Widal test is a serological test which is used for the diagnosis of enteric fever or typhoid fever. The test was developed by Greembaum and Widal in 1896. Typhoid or enteric fever is caused by a gram negative bacteria Salmonella enterica (Salmonella Typhi or Salmonella Paratyphi), found in the intestine of man. Salmonella paratyphi also causes Typhoid but of a milder form.

Salmonella possess O antigen on their cell wall and h antigen on their flagella. On infection, these antigen stimulates the body to produce specific antibodies which are released in the blood. The Widal test is used to detect these specific antibodies in the serum sample of patients suffering from typhoid using antigen-antibody interactions. These specific antibodies can be detected in the patient’s serum after 6 days of infection (fever).

Salmonella Typhi possesses O antigen on the cell wall and H antigen on flagella. Salmonella Paratyphi A and S. Paratyphi B also possess O antigen on their cell wall and but have AH and BH antigen on their flagella respectively.

Principle

 Widal test is an agglutination test in which specific typhoid fever antibodies are detected by mixing the patient’s serum with killed bacterial suspension of Salmonella carrying specific O, H, AH and BH antigens and observed for clumping ie. Antigen-antibody reaction. The main principle of Widal test is that if homologous antibody is present in patient’s serum, it will react with respective antigen in the suspension and gives visible clumping on the test slide or card.

Requirements

i) Fresh serum, stored at 2-8° Serum should not be heated or inactivated.

ii) The complete kit containing five vials containing stained Salmonella antigen

S. Typhi———-O antigen,

S. Tyhhi———- H antigen

S. Paratyphi —–AH antigen

S. Paratyphi —–BH antigen

iii) Widal positive control

iv) Widal test card or slide

v) Applicator stick

 

Procedure

1.      Bring all reagents to room temperature and mix well.

2.      Prepare 4 sets of test tubes for individual antigen. Each set contains 1- 8 tubes.

3.      Add 1.9 ml of 0.85% sterile saline to tube no. 1 of each antigen set.

4.      To tube no. 2-8 of all sets add 1 ml of physiological saline.

5.      To tube No. 1 of all sets add 0.1 ml of test sample to be tested and mix well.

6.      Transfer 1 ml of the diluted serum sample from tube No. 1 to tube No. 2 and mix well.

7.      Transfer 1 ml of the diluted serum sample from tube No. 2 to tube No. 3 and mix well. Continue this serial dilution till tube No. 7 in each set of antigen.

8.      Discard 1.0 ml of the diluted serum from tube No.7 of each set.

9.      So the dilutions of the serum sample from tube No. 1 to 7 respectively in each antigen set are 1:20, 1:40,1:80, 1:160, 1: 320, 1:640, 1: 1280.

10.  Tube no. 8 is negative control with 0.85% sterile saline.

11.              To one set i.e. from tube no.1- 8 add 50 µl of Salmonella typhi ‘O’ antigen.

12.  In second set i.e. from tube no.1- 8 add 50 µl of Salmonella typhi ‘H’ antigen.

13.  Respectively for third and fourth sets, add Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’ to all tubes from 1-8.

14.  Mix well, cover and incubate these tubes overnight at 37 degree Celcius (approximately 18 hours).

15.  After incubation dislodge the sediment and observe for agglutination.

Interpretaton :

The antibody titre of the test sample is its highest dilution that gives a visible agglutination. Agglutinin titre greater than 1:80 is considered as significant infection and low titres indicate absence of infection.