Widal-Tube method/ Titre slide method Principle, Techniques, and Interpretation

The Widal test is a serological test. It is used to diagnose enteric fever, including typhoid and paratyphoid fever. It is based on the specific antibodies (agglutinins) detection in the patient's serum against the bacteria Salmonella typhi and Salmonella paratyphi.

Principle: The Widal test is based on the principle of agglutination. The patient's serum is mixed with standardized suspensions of the specific Salmonella antigens O and Salmonella Antigen H of S. typhi for typhoid testing. Visible agglutination (clumping of bacteria) occurs if antibodies against these antigens are present in the patient's serum. This indicates a positive reaction. Salmonella paratyphoid antigen AH and Salmonella antigen BH are used for paratyphoid testing.

Techniques: The widal test is performed is performed in the following two ways

1. Widal slide agglutination test:

2. Widal tube agglutination test.

Widal slide agglutination test: It is a screening test. The sample showing a positive test is retested by the Widal tube agglutination test.

1. Preparation of Patient Serum: Collect the patient's blood sample using standard phlebotomy techniques. Centrifuge the blood sample to separate the serum from the cells. Transfer the clear serum to a clean, labeled tube. This serum contains the patient's antibodies.

2. Preparation of Agglutination Slides: Place clean microscope slides on a flat surface. Mark four rings on the slide. Label each ring as ‘O’. ‘H’ ‘AH’ and ‘BH’.

3. Mixing Samples: Mix each dilution of the patient's serum with the volume of saline solution (0.1mlserum:0.9ml saline). Add a drop of the diluted 50µl serum in each ring.

4. Performing the Test: Add a drop of the corresponding antigen in each ring. Mix the diluted serum and antigen drops by gently rocking the slide back and forth. Avoid shaking vigorously. Mix well to ensure uniform distribution of antibodies.

5. Incubation: Incubate the slide at room temperature for a specific period (usually 3 to 4 minutes). During this time, observe for any visible agglutination reactions.

6. Interpretation: Examine the slide under a microscope for agglutination. Agglutination appears as clumping or precipitation of the antigen-antibody complexes.

7. Reading Results: A positive reaction is indicated by agglutination observed. A negative reaction shows no agglutination.

8. Disposal: Dispose of all biohazardous materials and contaminated supplies according to institutional or regulatory guidelines.

Widal tube agglutination test.

1. Sample Collection: Blood is collected from the patient during the acute and recovery phases of the illness.

2. Serum Separation: The collected blood is allowed to clot. The serum is separated by centrifugation.

3. Preparation of Antigen Suspensions: Standardized suspensions of S. typhi and S. paratyphi antigens are prepared.

4. Serial Dilutions: The patient's serum is serially diluted to create a range of dilutions.

5. Mixing of Serum and Antigen: Each dilution of the patient's serum is mixed with the standardized bacterial antigens.

6. Incubation: The mixture is incubated at a specific temperature for a set period.

7. Observation: After incubation, the tubes or slides are examined for agglutination. Agglutination indicates the presence of antibodies.

Interpretation

1. Titration: The test is performed in a series of dilutions (1:20, 1:40, 1:80, etc.). The highest dilution showing agglutination is considered the endpoint. The result is reported as a titer, such as 1:80. It indicates the highest dilution showing agglutination.

2. Significance of Titers: A significant rise in titer between acute and recovery samples indicates an active infection. Single high titers (e.g., 1:160 or higher) suggest a recent infection or current carrier state.

Dr Pramila Singh