Introduction
The Widal tube test is a serological test used for the detection of Salmonella enterica antigens in the blood of a patient. This test helps in the diagnosis of typhoid fever, which is a severe bacterial infection caused by Salmonella enterica. The test is based on the principle of agglutination, which means that the antigen-antibody reaction leads to the formation of clumps or aggregates. In this blog post, we will discuss the principle of the Widal tube test, procedure, requirements, specimen, and observation.
Principle of Widal tube test:
The principle of the Widal tube test is similar to that of the Widal slide test. The test is based on the fact that when a patient is infected with Salmonella enterica, the body produces specific antibodies against the bacteria. These antibodies are known as O antibodies and H antibodies. The O antibodies are produced against the lipopolysaccharide (LPS) present on the outer membrane of the bacteria, while the H antibodies are produced against the flagellar protein (H antigen) present on the bacterial flagella.
The test involves mixing the patient’s serum with a suspension of Salmonella enterica antigens in a test tube. If the patient has been infected with Salmonella enterica, the antibodies in their serum will react with the bacterial antigens and cause clumping or agglutination. The agglutination reaction can be observed visually, and the titer or concentration of antibodies can be determined.
Requirements for Widal tube test
The following are the requirements for performing the Widal tube test:
- Clean and sterile glassware and equipment
- Salmonella enterica antigens
- Patient’s serum sample
- Microscope
- Incubator
- Reagents such as saline and distilled water
Specimen for Widal tube test
The specimen required for the Widal tube test is the patient’s serum. Blood is collected from the patient, and the serum is separated by centrifugation. The serum is then used for the test.
Procedure of Widal tube test:
The Widal tube test is performed using a tube agglutination technique. The procedure involves the following steps:
Step 1: Collect the patient’s blood sample and allow it to clot. Centrifuge the sample to obtain serum.
Step 2: Prepare a suspension of Salmonella enterica antigens in a test tube.
Step 3: Make a series of dilutions of the patient’s serum in separate test tubes.
Step 4: Add a small amount of the Salmonella enterica antigen suspension to each of the test tubes.
Step 5: Mix the contents of the test tubes using a vortex mixer or a shaker.
Step 6: Incubate the test tubes at 37°C for 18-24 hours.
Step 7: Examine the test tubes for agglutination.
process of Standard Tube Method:
To prepare for a Widal test, we would require the following objects:
- Patient’s serum
- O, H, AH and BH Antigens
- Normal Saline
- Pipette
- Test Tube Rack
- Test Tubes
- 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.
- First, take nine tubes and arrange them in the rack. In the case of O, mark the tubes in numbers from 1 to 9.
- 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.
- 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.
- 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.
- 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.
- Take a new (9th) test tube and add positive control.
- 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.
- 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.
- Now, mix well, cover and incubate the tubes at 37° C overnight (18 to 24 hours).
Which type of tube used in Widal test?
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.
Observation of Widal tube test:
The Widal tube test is observed visually for agglutination. The presence of clumps or aggregates indicates a positive reaction. The titer or concentration of antibodies can be determined by using different dilutions of the serum.
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:
- The results of the Widal test can be falsely positive in the case of past vaccination or S. Typhi infection.
- The Widal test is time-consuming; until a diagnosis is made, it becomes too late to start the treatment.
- A widal test can not distinguish between a patient’s past infection, current infection, or a S. Typhi vaccination.
- The test results can be falsely positive in typhus, acute falciparum malaria, chronic liver disease, rheumatoid arthritis, nephrotic syndrome and myelomatosis.
- 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.
