Widal test was developed by F Widal in 1896 and is a serological method for diagnosing enteric or typhoid fever (blood test for typhoid) caused by infection with pathogenic microorganisms such as Salmonella typhi, Salmonella paratyphi A, B and C.These microorganisms are transmitted to the human body by means of food and drinks that are contaminated with fecal matter.Early diagnosis and treatment are essential to avoid serious clinical complications.In the course of the infection, antibodies against Salmonella antigens are produced.The diagnostic test method is based on a visible agglutination reaction either in a test tube or on a slide between patient serum antibodies and antigens specifically prepared from Salmonella sp.
Principle of Widal test
A visible agglutination is formed during this test due to the reaction in a test tube or on a slide between antibodies present in the blood sample of the infected person and specific Salmonella antigens.
The organisms that cause enteric fever have two main antigens, namely somatic antigen, O and flagellar antigen, H and another surface antigen, Vi.In patients’ sera antibodies against Salmonella typhi O and H and Salmonella paratyphi AH and BH antigens are produced during the infection.Antigens specifically prepared from this organism are used in the agglutination test to detect the presence of antibodies in the sera of patients elucidated by these bacteria in response to infection.Some agglutinins are produced in the fever serum of the patient that react with somatic antigen O of Salmonella typhi, A or B of Salmonella paratyphi and then with flagellar antigen H common in most Salmonella species.Four specific antigen suspensions are used in this test which are H,Salmonella typhi O,Salmonella paratyphi- A,Salmonella paratyphi- B.
- If agglutination occurs with O antigen then Salmonella typhi is considered positive.
- When agglutination occurs in antigen A or B, it is confirmed to be positive for Salmonella paratyphi.
- Agglutination occurs in the H antigen circle for all cases of antigens such as O, A and B. Salmonella species are characterized by three antigens present on the cell as seen below
|O Antigen||This is a somatic antigen and is present in the outer cell membrane. The nature of the repeating units in the external O- polysaccharide chain determines its specificity. Somatic antigens are resistant to heat, alcohol and form compact and granular clumps when mixed with O antisera.|
|Vi antigens||It’s a virulence antigen that is a capsular polysaccharide overlaying the O antigen.This capsule is not necessary for infection but increases infectivity by making it less detectable by the body’s immune system.Vi antigen may interfere with testing of O antigen.|
|H Antigens||It’s a heat labile antigen that is inactivated by boiling and alcohol. H antigens quickly form fluffy clumps with the corresponding antisera. H antigen induces rapid antibody formation as it is strongly immunogenic.|
Materials and Reagents required for Widal test slide test
|Salmonella typhi ‘O’ Antigen||2-8° C|
|Salmonella typhi ‘H’ Antigen||2-8° C|
|Salmonella paratyphi ‘AH’ Antigen||2-8° C|
|Salmonella paratyphi ‘BH’ Antigen||2-8° C|
|Positive control||2-8° C|
|Negative control||2-8° C|
|Test Serum Sample||2-8° C|
|Disposable Mixing Sticks||RT|
The following should be taking into consideration before carrying out Widal test.
- Allow all reagents to reach room temperature before use
- Do not dilute any of the kit reagents
- Do not intermix the reagents.
- Do not freeze any of the kit reagents
- Ensure the glass slide is clean and dry prior to use.
- Always work with your personal protection equipments such as gloves,goggles etc
How to carry out Widal slide test
- As earlier mentioned,bring all reagents to room temperature and mix well.
- Mark the circles of slides as PC for Positive control, NC (Negative control), O, H, AH, BH as per antigen solutions used for testing
- Now add 1 drop of positive control (25μl) into the circle marked as PC of given glass slide.
- Then add 1 drop of negative control (25μl) into the reaction circle marked as NC.
- Add 1 drop of test sample (25μl) into each reaction circle labeled as O, H, AH, BH according to given antigen solution.
- Add 1 drop of Antigen solution of Salmonella typhi ‘H’ into PC and NC circle each.
- Mix well using new applicator stick for each circle.
- To circles labeled as O, H, AH, BH in which test samples has been added, add antigen solutions of Salmonella typhi ‘O’, Salmonella typhi ‘H’, Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’, respectively.
- Mix the content of each reaction circle uniformly with separate mixing stick.
- Rock the glass slide gently (approximately for one minute) and observe for agglutination.
Observation and Result of Widal Slide Test
After mixing the test sample with antigen solution, positive control, the negative control should be observed separately for the agglutination reaction.
Widal slide test Result Interpretation
During infection with Salmonella, the human body reacts to the antigenic stimulus and antibodies are produced.When the test sample is treated with colored and attenuated Salmonella antigen suspensions, the antibodies present in the sample react with the antigen suspension to produce a clearly visible agglutination visible to the naked eye.
How to carry out Widal tube test
- Bring all reagents to room temperature and mix well.
- Prepare 4 sets of test tubes for individual antigen. Each set contains 1- 8 tubes.
- Add 1.9 ml of 0.85% sterile saline to tube no. 1 of each antigen set.
- To tube no. 2-8 of all sets add 1 ml of physiological saline.
- To tube No. 1 of all sets add 0.1 ml of test sample to be tested and mix well.
- Transfer 1 ml of the diluted serum sample from tube No. 1 to tube No. 2 and mix well.
- 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.
- Discard 1.0 ml of the diluted serum from tube No.7 of each set.
- 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.
- 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 370 C (approximately 18 hours).
15. After incubation dislodge the sediment and observe for agglutination.
Observation and Result of Widal tube Test
After incubation, remove the sedimented button from each tube very carefully and observe the agglutination macroscopically. Note the titer of the antibody in the specified test sample for all antigens.
Widal tube test Result Interpretation
During infection with Salmonella, the human body reacts to the antigenic stimulus and antibodies are produced.When the test sample is treated with colored and attenuated Salmonella antigen suspensions, the antibodies present in the sample react with the antigen suspension to produce a clearly visible agglutination visible to the naked eye.The antibody titre of the test sample is the highest dilution that gives a visible agglutination.An agglutinin titre greater than 1:80 is considered a significant infection and low titres indicate the absence of infection.