Widal Test: Introduction, Principle, Test Requirement, Procedure

Introduction of Widal Test

Table of Contents

The Widal test is an agglutination test employed in the serological diagnosis of enteric fever. It is also applicable in febrile agglutinin tests. The test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist. Salmonella antibody starts appearing in the serum at the end of the first week and rises sharply during the third week of enteric fever. This test measures agglutinating antibody levels against O and H antigens.

Fig. Widal Test or Salmonella Agglutination Test Results-Positive and Negative

Principle of Widal Test

Patients suffering from enteric fever would possess antibodies in their sera that can react and agglutinate serial double-dilutions of killed, coloured Salmonella antigens in an agglutination test. Tests are available in the slide and tube agglutination method.

Requirements for Widal Test

Widal rack
Round-bottomed Felix tubes
Conical-bottomed Dreyer’s tubes
Water bath,
Doubly diluted patient serum in three-four rows,
Killed coloured suspensions of Salmonella Typhi O antigen, Salmonella Typhi H antigen, Salmonella Paratyphi A H antigen and Salmonella Paratyphi B H antigens
Specimen: For serological purposes, 1-3 ml of blood should be kept in a tube without anticoagulant for serum. A second sample, if possible, should be collected at the convalescent stage, at least 5 days later.

Salmonella Typhi O antigen, Salmonella Typhi H antigen, Salmonella Paratyphi A H antigen and Salmonella Paratyphi B H antigens and patient sera on tile prior to mixing for Widal Test — Fig. *Salmonella* Typhi O antigen, *Salmonella* Typhi H antigen, *Salmonella* Paratyphi A H antigen and *Salmonella* Paratyphi B H antigens and patient sera on tile prior to mixing for Widal Test

Preparation of Antigens

Salmonella Typhi 901 strain uses to prepare S. Typhi O and S. Typhi H antigens. O antigens for S. Paratyphi A and S. Paratyphi B can not take as they cross-react with S. Typhi O antigen. H antigen suspension prepares to treat overnight broth culture or saline suspension of Salmonella with 0.1 %formalin. For preparing O antigen suspension, Salmonella is growing on phenol agar (1:800) to inhibit flagella. The growth emulsifies in a small volume of saline, mixes with 20 times its volume of alcohol, heats at 40⁰C to 50⁰C for 30 minutes and centrifuges. The antigens treat with chloroform (preservative) and add appropriate dyes for easy identification of antigens.

Slide Widal Test

A slide Widal test is more popular among diagnostic laboratories as it gives rapid results.

Qualitative Widal Test Slide Method

Place one drop of each of the undiluted patients’ serum samples for the four antigens on the circled card. Similarly, add one drop of each of the four Salmonella antigens separately and gently rotate for one minute. The appearance of agglutination gives qualitative results. To know the titre for each of the antigens, repeat the test with dilutions of serum.

Quantitative Widal Test Slide Method

Place 80 μl, 40 μl, 20 μl, 10 μl and 5 μl of the patient’s serum each for the four antigens on the circled card. To each series of serum specimens, add one drop of a specific antigen to each, mixed and rotated for one minute. No agglutination in each of these. 80 μl corresponds to 1 in 20 dilutions, 40 μl to 1 in 40, 20 μl to 1 in 80, 10 μl to 1 in 160 and 5 μl corresponds to 1 in 320 titres.

Procedure-Tube Test

Dilute doubly patient serum by mixing and transferring from 1:10 to 1:640 in three-four rows. The first row usually comprises Felix tubes, where to add somatic S. Typhi O antigen. Take for all the remaining rows, Dreyer’s tubes; where add different flagellar H antigens. Each tube must contain 0.5 ml of diluted serum. Keep a test tube with only saline in each row as a control. Mix all the tubes (including the control) in a row with 0.5 ml of antigen suspension.

Treat the first row with S. Typhi O antigen, the second row with S. Typhi H antigen, the third row with S. Paratyphi AH antigen and the fourth row with S. Paratyphi BH antigen. Since infections by S. Paratyphi B are rare, usually omit this antigen in the test. After all the tubes have been treated with specific antigen suspensions, place the Widal rack in a thermostatically controlled water bath maintained at 37⁰C for overnight incubation. Another approach is to incubate the tubes at 50-55⁰C.

Reading the Widal Test Results

The control tubes must be examined first, where they should give no agglutination. The agglutination of O antigen appears as a “matt” or “carpet” at the bottom. Agglutination of H antigens appears loose, woolly or cottony. The highest dilution of serum that produces a positive agglutination is taken as the titre. The titres for all the antigens are noted.

Somatic (O) antigen (1:160): Positive-Salmonella Typhi
Positive control (PC)-Positive(agglutnation)
Negative control (NC)-Negative (no agglutination)

Interpretation of Widal Test

Timing of the test is important, as antibodies begin to arise during the end of the first week. The titres increase during the second, third and fourth week after which it gradually declines. The test may be negative in the early part of the first week. A single test is usually of not much value. A rise in titre between two sera specimens is more meaningful than a single test. If the first sample is taken late in the disease, a rise in titre may not be demonstrable. Instead, there may be a fall in titre. The baseline titre of the population must be known before attaching significance to the titres. The antibody levels of individuals in a population of a given area give the baseline titre.

Baseline titre in endemic areas

significant
O –antigen > 1:100
H –antigen > 1:200
Salmonella agglutinins are common among apparently healthy blood donors in Nepal with wide variation in baseline Widal agglutinin titre. Both O and H agglutinin titre >1:160 could be diagnostically significant in the presumptive diagnosis of enteric fever in Nepal. Patients already treated with antibiotics may not show any rise in titre, instead, there may befall in titre. They treated with antibiotics in the early stages may not give positive results. Patients who have received vaccines against Salmonella may give false-positive reactions. This can be differentiated from true infection by repeating the test after a week. True untreated infection results in rising in titre whereas vaccinated individuals don’t demonstrate any rise in titre. Those individuals, who had suffered from enteric fever in the past, sometimes develop anti-Salmonella antibodies during an unrelated or closely related infection.

This is termed an anamnestic response and can be differentiated from true infection by the lack of any rise in titre on repetition after a week. Antigen suspensions with fimbrial antigens may sometimes give false-positive reactions due to sharing of fimbrial antigens by some Enterobacteriaceae members. Antigen suspension must be devoid of fimbrial antigens. Titers raise in 2nd-week raise of titers diagnostic. Classically, a four-fold rise of antibodies in paired sera Widal test is considered diagnostic of typhoid fever. Moderate sensitivity and specificity 30% of culture-proven cases were found to be Widal negative ( WHO – TF guide).

False Positive and Negative Reactions with Widal Test

The Widal test may be falsely positive in patients who have had previous vaccination or infection with S. Typhi. Widal titers have also been reported in association with the hypogammaglobulinaemia of chronic active hepatitis and other autoimmune diseases. False-negative results may be associated with early treatment, with “hidden organisms” in bone and joints, and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic.

Limitation of Widal Test

The Widal test is time-consuming and often times when the diagnosis is reached it is too late to start an antibiotic regimen. In spite of several limitations, many physicians depend on the Widal test Classically, a four-fold rise of antibodies in paired sera Widal test is considered diagnostic of typhoid fever. However, paired sera are often difficult to obtain and specific chemotherapy has to be instituted on the basis of a single Widal test.

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Widal Test: Introduction, Principle, Test Requirement, Procedure, Result- Interpretation, and Limitations