WIDAL Test

WIDAL Test 

Aim

To detect Salmonella typhi  and Salmonella paratyphi  infection

Principle

WIDAL test is serological test for enteric fever.  Typhoid and paratyphoid fevers are grouped under Enteric fever.  typhoid is a life-threatening illness caused by Salmonella Typhi, usually transmitted through food and drinks contaminated with fecal matter. Salmonella paratyphi  causes paratyphoid fever.  The symptoms associated with the disease are high fever, fatigue, headache, abdominal pain, diarrhea or constipation, weight loss, and rashes.  

Widal Test is an agglutination test to detect the presence of serum agglutinins (H and O) in the serum of patients having typhoid or paratyphoid fever.  This is a reliable diagnostic test for typhoid fevers and was developed by Georges Ferdinand Widal in 1896.  Early diagnosis and treatment for this disease are essential to avoid serious clinical complications. Patients suffering from enteric fever will have antibodies in their sera which can react and agglutinate  stained Salmonella antigens in the reagent and gives visible clumping and agglutination either in a test tube or on a slide. The tube agglutination test is a quantitative method which can be used to determine titre values of antibodies present in the patient serum.

The antigens used in the test are H and O antigens of Salmonella typhi  and H antigen of S. Paratyphi A or S. Paratyphi B. O Antigen is somatic antigen present on the outer membrane of the bacterial cell. Somatic antigens are heat stable, alcohol resistant and forms compact and granular clumps when mixed with O antisera. H Antigens are flagellar antigens easily inactivated both by boiling and alcohol. H antigens form fluffy clumps when treated with the corresponding antisera.

Materials required

Salmonella antigen set

            Salmonella typhi  O Antigen

            Salmonella typhi  H Antigen

            Salmonella paratyphi  AH Antigen

            Salmonella paratyphi  BH Antigen

Positive control

Reaction slides and tubes

A.    Qualitative slide test

1.      All reagents were kept to thaw to room temperature

2.      The test glass slides provided in the test kit were cleaned and wiped

3.      One drop (0.05 ml) of undiluted serum and one drop of control serum were placed on separate circles on the slide

4.      After gentle shaking of the antigen vial, one drop of antigen O, H, AH and BH were added to the circle of the sample to be tested and also to the circle of control serum

5.      The drops were mixed using applicator sticks and spread over the entire circle area, and the slides were moved in a circular motion for 1 minute

6.      The slides were observed for agglutination

Observation

Agglutination was observed in first circle which indicated the presence of serum antibodies against O Antigen.

Result

As there was significant agglutination in circle where O antigen and serum was mixed, it indicates Salmonella infection.

B. Semi-quantitative slide test

1.      One drop of saline was added into the first circle in the slide and 5, 10, 20, 40, 80 ul of the test serum sample was added on the remaining circles.  

2.      To each circle, a drop of the antigen which showed agglutination with the test sample in the qualitative side test was added.

3.      Using separate applicator sticks, the contents of each circle were mixed uniformly over the reaction circles and the slides were moved in a circular motion for 1 minute

4.      The slides were observed for agglutination

Observation

Agglutination in the circle indicated the presence of serum antibodies against O Antigen. 

Result

The titre value of the antibody for the antigens were noted.  The antibody titre is highest dilution that gives a visible agglutination. Agglutinin titre greater than 1:80 is considered as infection and low titres indicate absence of infection.

C.    Quantitative tube test

1.          All reagents were kept to thaw to room temperature

2.          4 sets of test tubes were prepared for antigen.  Each set contained 1- 8 tubes

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

4.          1 ml of sterile saline was added to tube no. 2-8 of all antigen sets

5.          O.1 ml of test serum sample was added to tube No. 1 of all antigen sets and mixed well

6.          1 ml of the diluted serum sample was added from tube No. 1 to tube No. 2 and mixed well in all antigen sets

7.          1 ml of the diluted serum sample was transferred from tube No. 2 to tube No. 3 and mixed well and this serial dilution was continued till tube No. 7 in each set of antigen

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

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

10.      Tube no. 8 was kept as negative control.

11.      To one set of tubes (tube no.1- 8) 50 μl of Salmonella typhi  'O' antigen was added, to the second set of tubes, 50 μl of Salmonella typhi  'H' antigen was added and to the third and fourth sets of tubes, 50 μl of Salmonella paratyphi  'AH' and Salmonella paratyphi  'BH' antigens were added.

12.      All the tubes were mixed well, covered and incubated at 37^o C for overnight, approximately 18 hours

13.      After incubation the agglutination in the tubes were observed

Observation and Result

After incubation sedimented button in all tubes were dislodged gently and observed for agglutination.  The titre value of the antibody for all the antigens were noted.  The antibody titre is highest dilution that gives a visible agglutination. Agglutinin titre greater than 1:80 is considered as infection and low titres indicate absence of infection.