Widal Test is an agglutination test that detects the presence of serum agglutinins (H and O) in patients' serum with typhoid and paratyphoid fever.
When facilities for culturing are not available, the Widal test is reliable and valuable for diagnosing typhoid fevers in endemic areas.
The test was developed by Georges Ferdinand Widal in 1896.
The patient's serum is tested for O and H antibodies (agglutinins) against the following antigen suspensions (usually stained suspensions):
S. Typhi O antigen suspension, 9, 12
S. Typhi H antigen suspension, d
S. Paratyphi A O antigen suspension, 1, 2, 12
S. Paratyphi A H antigen suspension, a
S. Paratyphi B O antigen suspension, 1, 4, 5, 12
S. Paratyphi B H antigen suspension, b, phase 1
S. Paratyphi C O antigen suspension, 6, 7
S. Paratyphi C H antigen suspension, c, phase 1
Salmonella antibodies start appearing in serum at the end of the first week and rise sharply during the 3rd week of endemic fever.
In acute typhoid fever, O agglutinins can usually be detected 6–8 days after the onset of fever, and H agglutinins after 10–12 days.
It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a rising antibody titre.
Salmonella antigen suspensions can be used in both slide and tube techniques.
Principle of Widal Test
Bacterial suspension carrying antigen will agglutinate on exposure to antibodies to Salmonella organisms.
Patients suffering from enteric fever possess antibodies in their sera, which can react and agglutinate serial doubling dilutions of killed, colored Salmonella antigens in an agglutination test.
The main principle of the Widal test is that if the homologous antibody is present in the patient's serum, it will react with the respective antigen in the reagent, resulting in visible clumping on the test card and agglutination in the tube.
The antigens used in the test are "H" and "O" antigens of Salmonella Typhi and the "H" antigen of S. Paratyphi.
Paratyphoid "O" antigens are not employed as they cross-react with typhoid "O" antigen due to the sharing of factor 12.
"O" antigen is a somatic antigen, and "H" antigen is a flagellar antigen.
Preparation of Widal Antigens
H suspension of bacteria is prepared by adding 0.1 percent formalin to a 24-hour broth culture or saline suspension of an agar culture.
For the preparation of O suspensions of bacteria, the organisms are cultured on phenol agar (1:800) to inhibit flagella.
Standard smooth strains of the organism are used; S. Typhi 901, O and H strains are employed for this purpose.
The growth is then emulsified in a small volume of saline, mixed with 20 times its volume of alcohol, heated at 40°C to 50°C for 30 minutes, and centrifuged.
The antigens are treated with chloroform (preservative) and appropriate dyes are added for easy identification of antigens.
Procedure of Widal Test
SLIDE TEST
Place one drop of positive control on one reaction circle of the slide.
Pipette one drop of isotonic saline onto the next reaction circle as a 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 (positive control and negative control).
Add one drop each of ‘O’, ‘H’, ‘AH’, and ‘BH’ antigens to the remaining four reaction circles.
Mix the contents of each circle uniformly over the entire circle using separate mixing sticks.
Gently rock the slide back and forth and observe for agglutination macroscopically within one minute.
SEMI-QUANTITATIVE METHOD
Pipette one drop of isotonic saline into the first reaction circle.
Place 5, 10, 20, 40, and 80 µL of the test sample on the remaining circles.
Add one drop of the antigen, which showed agglutination with the test sample in the screening method, to each reaction circle.
Using separate mixing sticks, mix the contents of each circle uniformly.
Gently rock the slide back and forth and observe for agglutination macroscopically within one minute.
STANDARD TUBE TEST METHOD
In the Widal Test, two types of tubes were originally used:
Dreyer’s tube (narrow tube with conical bottom) for H agglutination
Felix tube (short round-bottomed tube) for O agglutination
Nowadays, 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 1.9 ml of isotonic saline into tube No.1 of all sets.
Add 1.0 ml of isotonic saline to each of the remaining tubes (2 to 8).
Add 0.1 ml of the serum sample to be tested into tube No.1 of each set 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 up to 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 sets serves as a saline control. The dilution of the serum sample achieved in each set is as follows:
Add one drop of the respective Widal Test antigen suspension (O, H, AH, and BH) from the reagent vials to all the tubes (1 to 8) of each set and mix well.
Cover the tubes and incubate at 37°C overnight (approximately 18 hours).
Gently dislodge the sedimented button and observe for agglutination.
Interpretation of Widal Test
SLIDE TEST
Agglutination is a positive test result; if observed with 20 µL of the test sample, it indicates the presence of clinically significant levels of the corresponding antibody in the patient's serum.
No agglutination is a negative test result, indicating the absence of clinically significant levels of the corresponding antibody in the patient's serum.
TUBE-METHOD
The titre of the patient serum using Widal test antigen suspensions refers to the highest dilution of the serum sample that shows visible agglutination.
A sample demonstrating a titre of 1:100 or higher for O agglutinations and 1:200 or higher for H agglutination should be considered clinically significant, indicating active infection. For example, in the above scenario, the titre is 160.
A diagnostic indication is the demonstration of a 4-fold rise between two samples.
H agglutination is more reliable than O agglutination.
Agglutinins typically start appearing in serum by the end of the first week, with a sharp rise in the second and third weeks. The titre remains steady until the fourth week, after which it declines.
Limitations of Widal Test
The Widal test, used to determine antibody titers, is time-consuming and often leads to delayed antibiotic treatment initiation.
Interpreting the Widal test should consider baseline titers in the local healthy population.
False positives may occur in individuals previously vaccinated or infected with S. Typhi.
The test cannot distinguish current infections from past infections or vaccinations.
Conditions like chronic active hepatitis and autoimmune diseases can affect Widal titers due to dysgammaglobulinemia.
False positives can also occur in typhus, acute falciparum malaria (especially in children), chronic liver disease with elevated globulin levels, rheumatoid arthritis, myelomatosis, and nephrotic syndrome.
False negatives may result from early treatment, "hidden organisms" in bones and joints, typhoid relapses, or poorly immunogenic infecting strains.
Early antimicrobial treatment or typhoid relapse can block antibody responses, leading to false negatives.
Severe hypoproteinemia can inhibit rises in O and H antibody titers.
Establishing a cutoff for baseline antibody levels in a specific area and community is challenging due to variations over time and across different regions.
In low typhoid endemic areas, weak and delayed O and H antibody responses limit the reliability of the Widal test.
Variations in test performance and interpretation among laboratories compromise its reliability.
The World Health Organization (WHO) advises caution in relying heavily on the Widal test for diagnosis due to these factors.
