28/03/2023
WIDAL TEST:
INTRODUCTION:
- Widal test is an agglutination test which detects the presence of antibodies in patient’s serum produced against the causative agents of enteric fever (Salmonella Typhi and Salmonella Paratyphi A, B and C).
- It is one of the common methods employed for serological diagnosis of typhoid and paratyphoid fever especially in the endemic regions.
PRINCIPLE:
- Individual infected with Salmonella Typhi or S. paratyphi produce antibodies against either somatic (O) antigens and/or flagellar antigens (H). These produced antibodies in serum, if exposed to bacterial suspension carrying homologous antigens, result in agglutination. The agglutination reaction can be seen as visible clumping.
- In the test, the patient’s serum is mixed with killed bacterial suspension of Salmonella carrying specific O, H, AH and BH antigens and observed for agglutination reaction. If the patient’s serum contains specific antibodies against the antigens, clumping is evident which indicates positive test. Absence of agglutination indicates a negative result.
REQUIRMENTS:
- Fresh serum,
- kit containing Salmonella antigen (O, H, AH and BH antigens),
- positive control,
- Widal test card or slide,
- Applicator stick.
PROCEDURE:
- Widal test can be done in two ways-
1. Rapid slide test: It may be qualitative or quantitative and performed on slide.
2. Tube test: It is performed in tubes, is quantitative and requires incubation. It is considered more reliable than slide test.
1. SLIDE TEST:
1. Place one drop of positive control on one reaction circles of the slide.
2. Pipette one drop of Isotonic saline on the next reaction cirlcle. (Negative Control).
3. Pipette one drop of the patient serum to be tested onto the remaining four reaction circles.
4. Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction circles. (PC & NC).
5. dd one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction circles.
6. Mix contents of each circle uniformly over the entire circle with separate mixing sticks.
7. Rock the slide, gently back and forth and observe for agglutination macroscopically within one minute.
2. TUBE METHOD:
1. Take 4 sets of 8 Kahn tubes/test tubes and label them 1 to 8 for O, H, AH and BH antibody detection.
2. Pipette into the tube No.1 of all sets 1.9 ml of isotonic saline.
3. To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
4. To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested and mix well.
5. Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
6. Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix well. Continue this serial dilution till tube no.7 in each set.
7. Discard 1.0 ml of the diluted serum from tube No.7 of each set.
8. 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 (control) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280.
9. To all the tubes (1 to 8) of each set add one drop of the respective WIDALTEST antigen suspension (O, H, AH and BH) from the reagent vials and mix well.
10. Cover the tubes and incubate at 37° C overnight (approximately 18 hours).
11. Dislodge the sedimented button gently and observe for agglutination.
RESULT:
1. POSITIVE:
Agglutination within a minute
2. NEGATIVE:
No agglutination indicative of absence of clinically significant levels of the corresponding antibody in the patient serum.
ADVANTAGES:
- It is a rapid test for screening enteric fever in endemic areas.
- When culture facilities are not available, Widal test is handy.
- It can detect infection caused by both Salmonella Typhi and Salmonella Paratyphi.
𝗡𝗼𝘁𝗲: H antigens indicate past infections while O antigen indicates current and active infection.
