- 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.
- The test was developed by Greembaum and Georges Ferdinand Widal in 1896 for diagnosis of enteric fever in humans.
- The Widal test utilizes antigen-antibody interactions to detect specific antibodies in the serum sample of typhoid patients. The antigens used are derived from the bacterial cells of Salmonella Typhi and Salmonella Paratyphi.
- Salmonella Typhi possesses O antigen on the cell wall and H antigen on flagella. Similarly, Salmonella Paratyphi A and Salmonella Paratyphi B also possess O antigen on their cell wall and but have AH and BH antigen on their flagella respectively.
- On infection, these antigens stimulate the body to produce specific antibodies which are released in the blood. These specific antibodies against the antigens can be detected in the patient’s serum after 6 days of infection (fever).
Objective of Widal Test
The test is performed to detect serum antibodies against Salmonella Typhi and Salmonella Paratyphi aiding in diagnosis of enteric fever in suspected patients.
Principle of Widal Test
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.
Requirements for Widal Test
Fresh serum, kit containing Salmonella antigen (O, H, AH and BH antigens), positive control, Widal test card or slide, Applicator stick.
Procedure of Widal Test
Widal test can be done in two ways-
- Rapid slide test: It may be qualitative or quantitative and performed on slide.
- Tube test: It is performed in tubes, is quantitative and requires incubation. It is considered more reliable than slide test.
- Place one drop of positive control on one reaction circles of the slide.
- Pipette one drop of Isotonic saline on the next reaction cirlcle. (Negative Control).
- Pipette one drop of the patient serum to be tested onto the remaining four reaction circles.
- Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction circles. (PC & NC).
- Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction circles.
- Mix contents of each circle uniformly over the entire circle with separate mixing sticks.
- Rock the slide, gently back and forth and observe for agglutination macroscopically within one minute.
- Pipette one drop of isotonic saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul of the test sample on the remaining circles.
- Add to each reaction circle, a drop of the antigen which showed agglutination with the test sample in the screening method.
- Using separate mixing sticks, mix the contents of each circle uniformly over the reaction circles.
- Rock the slide gently back and forth; observe for agglutination macroscopically within one minute.
STANDARD TUBE TEST METHOD
In Widal Test, two types of tubes were originally used:
(1) Dreyer’s tube (narrow tube with conical bottom) for H agglutination and
(2) Felix tube (short round-bottomed tube) for O agglutination.
Now a days 3 x 0.5 ml Kahn tubes are used for both types of agglutination.
- Take 4 sets of 8 Kahn tubes/test tubes and label them 1 to 8 for O, H, AH and BH antibody detection.
- Pipette into the tube No.1 of all sets 1.9 ml of isotonic saline.
- To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
- To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested and mix well.
- Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
- 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.
- Discard 1.0 ml of the diluted serum from tube No.7 of each set.
- 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.
- 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.
- Cover the tubes and incubate at 37° C overnight (approximately 18 hours).
- Dislodge the sedimented button gently and observe for agglutination.
Above protocols are obtained from WIDAL TEST: Swemed Diagnostics
Result Interpretation of Widal Test
Positive: Agglutination within a minute
Negative: No agglutination indicative of absence of clinically significant levels of the corresponding antibody in the patient serum.
- The sample which shows the titre of 1:100 or more for O agglutinations and 1:200 or more for H agglutination should be considered as clinically significant (active infection).
- Demonstration of 4-fold rise between the two is diagnostic.
- H agglutination is more reliable than O agglutinin.
- Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre remains steady till 4th week after which it declines.
- Rising antibody titre is more convincing evidence of infection than positive tests alone. It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a rising antibody titre.
- Low antibody titres are common in normal individuals and do not suggest an infection.
Advantages of Widal Test
- 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.
Limitations of Widal Test
- It is only a presumptive test and the diagnosis cannot be confirmed on the basis of Widal test alone.
- Test must be performed during a specified period only since tests done within 7 days of illness and after 4 weeks are usually negative.
- Since, low antibody titres are common in normal individuals, The Widal test should be interpreted in the light of baseline titers in a healthy local population.
- Quantitative tests can take a long time to complete.
- The Widal test may be falsely positive in patients who have had previous vaccination or infection with S.Typhi.
- False positive reaction may be common due to cross reactivity or in case of other non- Salmonella infections such as typhus, immunological disorders or chronic liver disease. Hence, more reliable methods of diagnosis such as blood culture is performed these days.
- Besides cross-reactivity with other Salmonellaspecies, the test cannot distinguish between a current infection and a previous infection or vaccination against typhoid.
- The World Health Organization (WHO) has said that due to the various factors that can influence the results of a Widal test, it is best not to rely too much on this test.
- Kenneth E.Sanderson, Shu-LinLiu1, LeTang and Randal N.Johnston.(2015).Salmonella Typhi and Salmonella Paratyphi A. Molecular Medical Microbiology (2 eds.). (2):1275-1306.
- Song JH, Park M, Na DS, Moon HB, Pai CH. Detection of Salmonella typhi in the blood of patients with typhoid fever by PCR. J Clin Microbiol 1993;31:1439-1443.
- Parry CM, Hien TT, Dougan G, White NJ, Farrar JJ. Typhoid fever. N Engl J Med. 2002 (347); 22:1770-1782.