WIDAL TEST : what it is, procedure and interpretation

Widal test is a serological test which is used for the diagnosis of enteric fever or typhoid fever. It was named so after the scientist who developed it. This test was first developed in 1896 by Greembaum and Widal.

Typhoid or enteric fever is a common problem in many populations affecting many children and adults and leading to serious complications including death. It is caused by a gram negative bacteria Salmonella enterica (which comes as either Salmonella Typhi or Salmonella Paratyphi) found in the intestine of man.

Salmonella possess O antigen on their cell wall and H antigen on their flagella. On infection, these antigen stimulates the body to produce specific antibodies which are released in the blood. The Widal test is used to detect these specific antibodies in the serum sample of patients suffering from typhoid using antigen-antibody interactions.

These specific antibodies can be detected in the patient’s serum after 6 days of infection (fever). Salmonella Typhi possesses O antigen on the cell wall and H antigen on flagella. On the other hand, Salmonella Paratyphi A and S also possess O antigen on their cell wall and but have AH and BH antigen on their flagella respectively.

Principle of Widal Test

Widal test is an agglutination test in which specific typhoid fever antibodies are detected by mixing the patient’s serum with killed bacterial suspension of Salmonella. These bacteria suspension carry specific O, H, AH and BH antigens and after mixture, they are observed for clumping (antigen-antibody reaction).

The main principle behind Widal test is that if homologous antibody is present in patient’s serum, it will react with respective antigen in the suspension and give visible clumping on the test slide or card.

Requirements For Widal Test

Here are the things needed to conduct a Widal test.

  • Fresh serum, stored at 2-8°. The serum should not be heated or inactivated.
  • The complete kit containing five vials of stained Salmonella antigen

S. Typhi———-O antigen
S. Tyhhi———- H antigen
S. Paratyphi —–AH antigen
S. Paratyphi —–BH antigen

  • Widal positive control.
  • Applicator stick
  • Widal test card or slide.

Procedure of Widal Test

Widal test can be done in two ways. One is rapid test on slide and another is tube test in which result may be obtained after one night of incubation.

For Rapid Slide Test

  1. Clean the glass slide or test card supplied in the kit well and make it dry.
  2. Label the circles (1, 2, 3, 4, 5 and 6) in the test card as O, H, AH, BH, Negative control and Positive control).
  3. Place a drop of undiluted test serum in each of the four labelled circle (1, 2, 3 and 4) ie O, H, AH and BH and place a drop of Negative control serum in circle 5 and Positive control in circle 6.
  4. Add a drop of antigen O, H, AH and BH in circle 1, 2, 3, and 4 respectively and no antigen in circle 5 and O/H antigen in circle 6.
  5. Mix the content of each circle with a separate wooden applicator stick and spread to fill the whole area of the individual circle.
  6. Rock the test card for a minute and observe for agglutination.

If agglutination is visible within 1 minute, proceed for quantitative slide test or tube test for the quantitative estimation of the titre of the antibody.

For Qualitative Test

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.

For Quatitative Test

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 1in 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.

Result Interpretation And Considerations

Low titres are often in normal individuals. A single positive is less significant than the rising antibody titre, since rising titre is considered to be a definite evidence of infection.
Any antibody titre greater than 1 : 80 is considered significant and usually suggests positive test for Salmonella infection.

A titre of 100 or more for O antigen is considered significant and a titre in excess of 200 for H antigens is considered significant. 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.

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