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Widal Test Principle, Procedure, Result, Advantages, Limitation

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Widal Test

  • Widal test is an Agglutination testing test that detects antibodies in the serum of the patient which are generated by the cause of the febrile illness of the enteric (Salmonella Typhi and Salmonella paratyphi B A, as well as C).
  • It is one of the most frequently utilized methods to identify the causality of typhoid or paratyphoid illness especially in the areas that are endemic.
  • The test was developed by Greembaum and Georges Ferdinand Widal in 1896 to determine the presence of the presence of enteric fever in humans.
  • The Widal test uses interaction between antigens and antibodies to determine particular antibodies present in the serum from patients with the typhoid. The antigens that are tested come from bacteria that cause Salmonella Typhi and Salmonella Paratyphi.
  • Salmonella Typhi possesses O antigen on its cell wall in addition to H antigens that are on its flagella. Similar to this, Salmonella Paratyphi A and Salmonella Paratyphi B also contain O antigens that line their cell wall. however, they do have AH as well as BH antigens in their flagellas, respectively.
  • In the event of an infection, these antigens trigger the body to produce specific antibodies that are released into the blood. Specific antibodies generated against antigens are recognized by the serum levels of patients within the first 6 days following the onset of the illness (fever).

The Objective of Widal Test

The test will identify the presence of antibodies in serum against Salmonella Typhi and Salmonella Paratyphi aiding in diagnosing the presence of enteric fever in those who are suspects.

Principle of Widal Test

People infected by Salmonella Typhi or S. paratyphi develop antibodies in the form of somatic (O) antigens or flagellar antigens (H). The antibodies that are produced in serum when exposed to bacterial suspensions containing homologous antigens, cause the formation of agglutination. The agglutination process is visible as clustering.

For the testing, patient’s blood is mixed with dead suspensions of bacterial strains from Salmonella that contain specific O H AH as well as BH antigens and analyzed for agglutination reactions. If the patient’s serum has specific antibodies to the antigens, it will show clumping which is a sign of positive results. Agglutination in absence is a sign of a negative test.


Requirements for Widal Test

Clean and fresh serum kit with Salmonella antigen (O H, O antigens for AH and BH) Positive control Test card for Widal, or the slide applicator stick.

Procedure of Widal Test

The Widal test can be conducted in two methods:

  1. Rapid slide test: Rapid slide test may be qualitative or quantitative and is conducted on slides.
  2. Tube test: It’s conducted in tubes, is quantifiable and requires an incubation period. It’s believed to be more precise than slide tests.


  1. Place the equivalent of 1 drop Positive Control on any of the circles that react on your slide.
  2. Pipette one drop Isotonic salicylate onto the next circular ring of the reaction. (Negative Control).
  3. Pipette the drop of serum from the patient for testing using the 4 circles.
  4. Be sure to include one drop of Test Antigen Solution “H within both circles. (PC & NC).
  5. Make sure you add one drop each O”and “H “AH” as well as “BH” antigens in each of the remaining four reaction circles.
  6. Combine the content of the circle in a uniform way across the entire circle using various mixing tools.
  7. Gently slide between left and right, then search for macroscopically Agglutination that takes less than a minute.


  1. Pipette one drop Isotonic solution onto the initial circle of reaction Then, put 5, 10, 20 and to 80ul from the testing specimen on the remaining circles.
  2. Include in each circle the amount of antigen that was agglutinated to the test sample, using the screening process.
  3. With separate mixing sticks, Mix the ingredients from each circle uniformly across the reaction circles.
  4. Slide the slides gently between the slides. Watch for the macroscopically visible agglutination within less than a minute.


The Widal Test, two types of tubes were first used:

(1) Dreyer’s Tube (narrow tube having a conical top) for H agglutination as well as

(2) Felix tube (short round-bottomed tube) for O Agglutination.

Nowadays three x 0.5 milliliter Kahn tubes are utilized to treat various types of Agglutination.

  1. Take four sets of eight Kahn tubes/test tube and label them with numbers 1 through 8. This will allow for O H, AH and identification for BH antibodies.
  2. Pipette into the tube No.1 of all sets 1.9 Ml of isotonic sodium.
  3. For each tube (2 through 8) In each of the remaining tubes (2 to 8) add 1.0 mg of sodium isotonic.
  4. In the No.1 tube In each row, in each row, add 0.1 milliliters of the sample of serum for testing, and mix well.
  5. Transfer 1.0 milliliters of dilute serum from tube no.1 into the tube no.2 Mix thoroughly.
  6. Transfer 1.0 milliliters of the dilute quantity from the tube no.2 to tube no.3 and mix thoroughly. Repeat this dilution series until you reach tubes no.7 in each sequence.
  7. Remove 1.0 milliliters (milliliters) of liquid dilute in tubes No.7 in each of the sets.
  8. Tube No.8 in all sets functions as a saline control. The dilution of the serum sample taken in each set is as follows: Tube No. 1 2 3 4 5 6, 7 (control) Dilutions 1;20 1;40 1:80 1:160 1;320 1:1280 1.
  9. Within all the tubing (1 up to 8.) of each set you should put in one drop of WIDALTEST suspension (O H, O, AH and H) in the vials of reagent , and mix well.
  10. The tubes should be covered with lids, and allow them to incubate for 37 degrees C for the duration of an entire night (approximately around 18 hours).
  11. Take the button off which has been swallowed into the sand and look for an Agglutination.

These protocols can be accessed through the WIDAL TEST. Swemed Diagnostics

Result Interpretation of Widal Test

Positive:  Agglutination occurs within one minute

Negative: No Agglutination, in that sense there aren’t clinically significant levels of the antibody that is relevant within the blood of the subject.

  • The specimen that has an ratio of 1:100 or more for O Agglutinations, or 1:200 or more for H agglutination, is considered to be significant medically pertinent (active condition).
  • The observation of a fourfold increase in both cases indicates the diagnosis.
  • Agglutination with H is more stable than O agglutinin.
  • Agglutinin is first visible in serum at the end in the beginning of week and then increases dramatically during the 2nd and 3rd weeks , and the amount stays steady up to the 4th week after which it begins to decrease.
  • The rise in the amount of antibodies is more reliable as a sign for the existence of infection than positive tests. It is suggested to test two samples of sera over a period of 7 , 10, and 14 days in order to detect a rise in antibody levels.
  • Low levels of antibody are common in healthy individuals, however, they is not an indication of an infection.

Advantages of Widal Test

  • It’s a fast test to determine the presence of enteric fever in regions with the highest incidence.
  • If culture facilities aren’t readily accessible Tests using Widal are helpful.
  • It is able to identify diseases due to Salmonella Typhi and Salmonella Paratyph.

Limitations of Widal Test

  • The Widal test can be lengthy (to detect the level of antibody) and, most of the time once the the diagnosis has been confirmed, it’s for too late to initiate an antimicrobial treatment.
  • The Widal test should be viewed in relation to test results for a healthful local populace.
  • The Widal test is not positive for patients who have had previous vaccination or who have acquired S. Typhi.
  • Additionally, cross-reactivity can occur in other Salmonella species, it is not possible to differentiate between an ongoing illness and an infection that was present before or an earlier vaccine against Typhoid.
  • Widal titers have also been reported in association with the dysgammaglobulinaemia of chronic active hepatitis and other autoimmune diseases.
  • False negative Widal testing results have been seen in acute falciparum and typhus as well as malaria
  • (particularly for (especially for children) The chronic liver disease (particularly in children) is characterised by increased levels of globulin and conditions such as rheumatoid arthropathy myelomato and nephrotic disorder.
  • False negatives could result from an early diagnosis of “hidden bacteria” in bone and joints or after relapses with the typhoid infection. In some instances the strains that are infected aren’t immune-friendly.
  • False negative Widal tests can result from antibodies which are blocked by early antimicrobial therapy , or following a rebound of typhoid.
  • Hypoproteinaemia severe can trigger the increase in H antibody levels and the zero.
  • The amount of antibody is found in healthy individuals however, they may differ from time to time and between different regions, which makes it difficult to establish the minimum level of the baseline antibody in the community or area.
  • In areas where typhoid infection is not an endemic disease, the in the area, slow or weak O and H antibodies can reduce the efficacy of testing using tests using the Widal test. Additionally, there are variations between labs regarding the interpretation and results of Widal tests that affect the accuracy of the test.
  • The World Health Organization (WHO) has declared that due to many variables that could influence the results from Widal, there are many factors that could affect the results of Widal test, it is recommended not to rely too heavily on the outcome of this test.


  • https://en.wikipedia.org/wiki/Widal_test
  • https://www.testing.com/tests/widal-test/
  • https://www.portea.com/labs/diagnostic-tests/widal-test-typhoid-test-typhidot-124/
  • http://www.ncbi.nlm.nih.gov/pubmed/7342378
  • https://cvi.asm.org/content/19/11/1833
  • http://www.downtoearth.org.in/news/10minute-typhoid-test-38903
  • http://www.ncbi.nlm.nih.gov/pubmed/12592993
  • http://www.cochrane.org/CD008892/INFECTN_accuracy-rapid-diagnostic-tests-detecting-typhoid-and-paratyphoid-enteric-fever


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