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Widal Test - Introduction, Principle and Procedure




A Widal test meaning is a sersology blood test that helps detect typhoid or enteric fever in the body. This test was first conducted in 1896 by Georges Ferdinand Widal and was named after him. 

The Widal test is an advanced way to check for antibodies that your body makes against the salmonella bacteria that causes typhoid fever. It looks for O and H antibodies in a patient’s sample blood (serum).

This test helps detect life-threatening illnesses like typhoid fever. However, interpreting the Widal test is important to arrive at the correct results. 

Keep reading this blog to understand the meaning of the Widal test, its principle, procedure, and test result interpretation.

Typhoid Fever and Widal Test

Typhoid fever, also known as enteric fever, is a severe illness caused by salmonella bacteria. This bacteria affects your digestive system after you consume food or water contaminated with faecal material. 

A person with typhoid fever will experience symptoms like fatigue, high fever, headache, diarrhoea or constipation, abdominal pain, weight loss, and red spots. It is crucial to detect and treat typhoid fever as soon as possible to prevent further complications like severe intestinal bleeding or perforation.

There are various tests available to detect typhoid fever, such as stool culture, bone marrow, etc. One such test is the Widal test, which detects the presence of antibodies that your body makes against salmonella bacteria during typhoid.

The bacteria that causes typhoid fever is Salmonella bacteria. It can spread from person to person or from consuming contaminated food. The bacteria are of two types:

  1. Salmonella Typhi, also known as S. Typhi,
  2. Salmonella Paratyphi, which is also known as S. Paratyphi

Now, the bacteria S. Typhi has two antigens named:

  1. S. Typhi O (TO), primary antigen
  2. S. Typhi H (TH), secondary antigen

On the other hand, the bacteria S. Paratyphi has the following two antigens:

  1. S. Paratyphi A
  2. S. Paratyphi B 

To understand and interpret the Widal test reports, we will discuss these values later in this blog to understand and interpret the Widal test reports.

Principle of Widal Test

When you consume food contaminated with salmonella bacteria, it enters your body in the form of antigens. After reaching your digestive system, your immunity creates antibodies against those antigens. As a result, antigens agglutinate (react), and typhoid fever is detected.

This agglutination test detects the antibodies your body has made in response to a particular bacteria or antigen. If you have typhoid fever, your sera (blood) will possess antibodies that will react and agglutinate salmonella antigens in an agglutination test. 

The main Widal test principle is that if a particular antibody is present in the serum, it will react with a specific antigen and show visible clumping on the test card. 

The following step of this test measures the titre of the positive antigen:

Preparation of Widal Test

Slide Widal Test

To prepare for a Widal test, we would require the following objects:

  1. Patient’s serum
  2. Pipette (lab tool)
  3. Serum
  4. S. Antigen ( O, H, AH, BH)
  5. Slide
  6. Mix Stick
  7. Stopwatch

You will also need reagents to detect antigens like O Ag (somatic or surface Ag), H Ag (flagella), AH Ag, BH Ag, etc. 

The Widal test procedure is carried out in two steps:

  1. Qualitative Widal Test

For this test, you will use a slide with 6 reaction circles, marked as O, H, AH, BH, PC and NC. To begin with,

  1. Put one drop of the patient’s serum in four reaction circles, i.e., O, H, AH, BH.
  2. Add one drop of positive control in the PC circle and one in the NC circle.
  3. Next, add one drop of O antigen in the O circle, P antigen in the P circle, AH antigen in the AH circle, and BH antigen in the BH circle, respectively.
  4. Add any antigen, i.e., O, H, AH, BH in both PC and NC.
  5. Next, mix the serum and antigen in each circle properly so that the mixture doesn’t go out of the circle and touch the slide.
  6. Also, one mixture should not mix with another, as it can influence the test results.
  7. Finally, rotate the slide in a slow circular motion to ensure a proper mixture of serum and reagent.

Once everything is done, you can see the results. If the test is positive, the test will be similar to the PC (+ve control circle), and if it’s negative, it will be similar to NC (-ve control circle). In other words, if there is any agglutination, the test results will be positive and vice versa. 

Now, if the test results are positive, the next stage will involve a quantitative test. To confirm the diagnosis of typhoid fever, we will take the reagent of the antigen that is positive. For example, if O was positive in the qualitative test, we will take the O reagent in the quantitative test. 

In most cases, O and H, i.e., S. Typhi are used to confirm the diagnosis. Only a few people have their AH or BH positive during a qualitative test.

2. A quantitative Widal test: It is a semi-procedure test, which means you will check for O if O is positive, H if H is positive, and both if both are positive. To perform this test, follow these steps:

  1. Use a different slide with 8 circles: four for O antigen and four for H.
  2. Now, if O was positive in the last test, put 5 ul patient’s serum in the 1st O circle, 10 ul in the 2nd, 20 ul in the 3rd, and 40 ul in the 4th circle horizontally.
  3. In the same way, put one drop of the specific reagent in all four circles. 
  4. To report, mark the values from the right side. Mark 1:40 on the fourth circle, 1: 80 on the 3rd circle, 1:160 on the 2nd, and 1:320 on the 1st circle.

Interpretation of Widal Test-Slide Method

After mixing the serum and reagent properly and rotating the slide, wait for the results to show. The result will be positive if it shows positive in more than 100 in the O circle and 200 in the case of H.

In other words, it is a positive Widal test if the titre is above 100 in O and 200 in H. However, the results can be negative due to antibiotics, malaria, dengue, or the fever has affected you for more than a few days, etc.

You can interpret your test results in the following ways:

  • Negative if S. Typhi is smaller than or equal to 1:80 and positive if S. Typhi is more than or equal to 1:160.
  • Negative if S. Paratyphi is smaller than or equal to 1:80 and positive if S. Paratyphi is more than or equal to 1:160.

If S. Typhi O is positive, then you have an active fever. However, in the case of positive S. Typhi H, the fever is a past infection. Moreover, the Widal test normal range is when the titre is equal to or below 1:80 in both O and H antigens.

Tube Widal Test

Apart from qualitative and quantitative Widal tests, there is another test method; the standard tube method. Here, eight tubes are taken, diluted and observed. The first test can screen for typhoid fever; however, a tube Widal test effectively confirms a fever.

Originally, two types of tubes were used to detect typhoid fever:

  • Dreyer’s tube for H agglutination
  • Felix tube for O agglutination

Nowadays, 3 x 0.5 ml Kahn tubes are used for both O and H agglutination.

Process of Standard Tube Method:

To prepare for a Widal test, we would require the following objects:

  1. Patient’s serum
  2. O, H, AH and BH Antigens
  3. Normal Saline
  4. Pipette
  5. Test Tube Rack
  6. Test Tubes
  7. Water Bath

Before beginning this test, it is essential to note that the tube method is a dilution technique that has to be done correctly. Let’s understand this test in a simpler language.

  1. First, take nine tubes and arrange them in the rack. In the case of O, mark the tubes in numbers from 1 to 9.
  2. Add and mix 0.1 ml normal saline and 0.9 ml serum in the first test tube. On the other hand, add 0.5 normal salines to each remaining tube.
  3. Next, take 0.5 ml from the 1st tube and add in the second. This will result in 0.5 ml remaining solution in test tube 1 and 1 ml in test tube 2nd.
  4. Repeat this process i.e., take 0.5 ml from the last tube and add it to the next tube to make it 1 ml. With the 8th tube, take 0.5 ml and keep that in another separate tube.
  5. Mix all the tubes properly. This will give us primary serial dilution of all the tubes from 1st to 8th as 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280, respectively.
  6. Take a new (9th) test tube and add positive control.
  7. Next, add 0.5 ml of respective antigen (O, H, AH, BH) in all eight tubes. This will make the final volume of each tube 1 ml.
  8. After adding a reagent to all the tubes, we will have the final serial dilution of all the tubes from 1st to 8th as 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2580.
  9. Now, mix well, cover and incubate the tubes at 37° C overnight (18 to 24 hours).

Interpretation of Widal Test-Tube Method

If you have typhoid fever and there is some agglutination, you will see that the 9th tube (positive control) will look similar to one of the eight other tubes. If there is no enteric fever, there will be no change in the normal range of the eight tubes and widal test.

If the tube that showed agglutination has a titre of more than 1:100 in case of O and 1: 200 in H, it will be widal test positive (active infection). Other than this, rest titers are considered the normal range of a widal test.

Limitations of Widal Test

No doubt, the widal test is a quick and effective way to diagnose typhoid fever, but it has some limitations as well, including:

  1. The results of the Widal test can be falsely positive in the case of past vaccination or S. Typhi infection.
  2. The Widal test is time-consuming; until a diagnosis is made, it becomes too late to start the treatment.
  3. A widal test can not distinguish between a patient's past infection, current infection, or a S. Typhi vaccination.
  4. The test results can be falsely positive in typhus, acute falciparum malaria, chronic liver disease, rheumatoid arthritis, nephrotic syndrome and myelomatosis. 
  5. Because so many factors can influence the test results, it is better to not just depend on this test for typhoid diagnosis.

However, it is always best to choose trustable labs regarding screening. A delay or ineffective screening can cause a delay in the treatment.

Price of Widal Test

A widal test is a quick and affordable test which is mostly used in developing countries where typhoid fevers are prevalent. The widal test cost can differ based on location, lab centre and other criteria. With Metropolis Labs, the cost of the widal test is just xxx. 

Our website features allow you to track your sample, download reports, book a home visit, upload prescriptions, etc. Contact metropolis labs today to book a widal test and get results quickly.


A Widal test is a quick procedure to diagnose typhoid fever or enteric fever. It only requires a patient's serum and some reagents to detect this fever. However, because different factors can influence the test results, it is always best to perform this test one week after the infection, following proper instructions. 

If you have typhoid fever or are experiencing its symptoms, you can book a Widal test today.

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