Widal test: Introduction, procedure, result Interpretation and limitations

Widal test

 Introduction of Widal test

Widal test is an agglutination test employed in the serological diagnosis of enteric fever. It also applicable in  febrile agglutinins 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 first week and rises sharply during the third week of enteric fever. This test measures agglutinating antibody levels against O and H antigens.

Principle of WIDAL test

Patients’ suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial double-dilutions of killed, colored Salmonella antigens in a agglutination test. Tests are available in 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 colored suspensions of Salmonella Typhi O antigen, Salmonella Typhi H antigen,Salmonella  Paratyphi A H antigen and Salmonella Paratyphi B H antigens


    For serological purposes, 1-3 ml of blood should be kept into a tube without anticoagulant for serum. A second sample, if possible, should be collected at the convalescent stage, at least 5 days later.

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  treating overnight broth culture or saline suspension of Salmonella with 0.1%
formalin. For preparing O antigen suspension, Salmonella are growing on phenol agar (1:800) to inhibit flagella. The growth  emulsifies in 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

One drop each of undiluted patients’ serum samples for the four antigens are placed on the circled card and one drop of each of the four Salmonella antigens are added separately and gently rotated for one minute. Appearance of agglutination gives qualitative results. To know the titre for each of the antigens, the test is repeated with dilutions of serum.

Quatitative  Widal test slide method

80 μl, 40 μl, 20 μl, 10 μl and 5 μl of patient’s serum each for the four antigens are placed on the circled card. To each series of serum specimen, one drop of specific antigen is added to each, mixed and rotated for one minute. Agglutination in each of these is noted. 80 μl corresponds to 1 in 20 dilution, 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 titre.

Procedure :- Tube test

Patient serum is doubly diluted by mixing and transferring from 1:10 to 1:640 in three-four rows. First row usually comprises of Felix tubes, where somatic S.typhi O antigen is added. For all the remaining rows, Dreyer’s tubes are taken; where different flagellar H antigens are added. Each tube must contain 0.5ml of diluted serum. A test tube with only saline is kept in each row as control. All the tubes (including control) in a row are mixed with 0.5ml of antigen suspension. The first row is treated 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, this antigen is usually omitted in the test. After all the tubes have been treated with specific antigen suspensions, the Widal rack is placed 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 titre. The titres for all the antigens are noted.

Interpretation of Widal test

Timing of test is important, as antibodies begin to arise during end of first week.The titres increase during second, third and fourth week after which it gradually declines.The test may be negative in early part of first week. 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. 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 be fall in titre. Patients 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 rise 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 anamnestic response and can be differentiated from true infection by 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 antibody in paired sera Widal test is considered diagnostic of typhoid fever. Moderate sensitivity and specificity 30% of culture proven cases 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 dysgammaglobulinaemia 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 diagnosis is reached it is too late to start an antibiotic regimen. In spite of several limitation many physicians depend on Widal test Classically, a four-fold rise of antibody 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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