Widal Test- Principle, Procedure, Types, Results, Uses

Widal test is a serological test that is used to diagnose enteric fever, also known as typhoid or paratyphoid fever, caused by the bacteria Salmonella Typhi and Paratyphi. Enteric fever is a systemic infection that can cause high fever, abdominal pain, headache, diarrhea, and sometimes complications such as intestinal perforation or hemorrhage. It is transmitted by ingestion of contaminated food or water.

Widal test detects the presence of antibodies against specific antigens of Salmonella Typhi and Paratyphi in the blood serum of the patient. Antibodies are proteins produced by the immune system in response to foreign substances (antigens) that enter the body. Salmonella Typhi and Paratyphi have two types of antigens: O (somatic) and H (flagellar). O antigens are present on the cell wall of the bacteria, while H antigens are present on the flagella, which are hair-like structures that help the bacteria move.

Widal test is based on the principle of agglutination, which is a visible clumping of antigens and antibodies. When a serum sample containing antibodies against Salmonella Typhi or Paratyphi is mixed with a suspension of killed and stained antigens of these bacteria, an agglutination reaction occurs if there is a match between the antibodies and the antigens. The degree of agglutination indicates the level of antibodies in the serum.

Widal test was developed in 1896 by Georges-Fernand Widal, a French physician and bacteriologist. He originally described this test to diagnose Salmonella Paratyphi B infection. Later it was modified and used to diagnose all types of enteric fever.

Widal test is widely used throughout the world as a major diagnostic tool for detecting enteric fever, especially in areas where culture facilities are not available or reliable. However, Widal test has some limitations, such as false-positive or false-negative results, cross-reactivity with other infections or vaccinations, and variation in antibody levels depending on the stage and duration of infection. Therefore, Widal test results need to be interpreted carefully in conjunction with clinical symptoms, history of exposure, and other laboratory tests.

The objective of the Widal test is to detect the presence of serum antibodies against Salmonella Typhi and Paratyphi, which are the causative agents of enteric fever (also known as typhoid or paratyphoid fever). Enteric fever is a serious systemic infection that can cause high fever, abdominal pain, diarrhea, and complications such as intestinal perforation and hemorrhage. It is endemic in many developing countries where sanitation and hygiene are poor, and it can also be acquired through travel or foodborne outbreaks. The diagnosis of enteric fever is important for initiating appropriate antibiotic therapy and preventing further transmission of the disease.

The Widal test is a serological test that measures the level of agglutinating antibodies against the O (somatic) and H (flagellar) antigens of Salmonella Typhi and the AH and BH antigens of Salmonella Paratyphi A and B. These antigens are immunogenic and stimulate the production of specific antibodies in the host`s immune system after 6-8 days of infection. The Widal test is based on the principle of antigen-antibody agglutination reaction, which means that when a homologous antibody reacts with a specific insoluble antigen in the presence of a suitable electrolyte and at optimal temperature and pH, a visible clumping or agglutination occurs. The Widal test can be performed by two methods: rapid slide agglutination test and tube agglutination test. The slide method is simpler and faster, but less sensitive and accurate than the tube method. The tube method can also measure the antibody titer, which is the highest serum dilution that gives a positive reaction.

The Widal test is widely used throughout the world as a major diagnostic tool for detecting enteric fever, especially when facilities for culturing are not available or when the culture results are delayed or negative. However, the Widal test also has some limitations, such as false-positive results due to previous infection or vaccination, cross-reactivity with other infections, low sensitivity and specificity in endemic areas, variation in antigen quality and antibody titer cutoffs, and interference by antibiotic treatment or immune disorders. Therefore, the Widal test should be interpreted with caution and in conjunction with clinical symptoms, epidemiological data, and other laboratory tests.

Widal test is based on the principle of antigen-antibody reaction of agglutination type. Agglutination is the visible clumping of antigen and antibody molecules that occurs when they bind to each other. The antigen and antibody molecules must be homologous, meaning they have complementary shapes and charges that allow them to fit together.

In the case of Widal test, the antigens are the O and H antigens of Salmonella Typhi and the AH and BH antigens of Salmonella Paratyphi A and B, respectively. The O antigens are located on the cell wall of the bacteria, while the H antigens are located on the flagella. The AH and BH antigens are specific types of H antigens found only in Salmonella Paratyphi A and B. The antibodies are the IgM and IgG antibodies produced by the host`s immune system in response to the infection by Salmonella.

The Widal test involves mixing the patient`s serum (which contains antibodies) with commercially prepared antigens (which are insoluble particles coated with bacterial antigens) in a suitable medium (such as saline solution) at an optimal temperature and pH. If the serum contains antibodies that match the antigens, they will bind to them and form visible clumps (agglutination). The degree of agglutination indicates the level of antibodies in the serum, which reflects the severity and stage of the infection.

The Widal test can be performed by two methods: slide agglutination test and tube agglutination test. The slide agglutination test is a rapid and simple method that can be done on a glass slide or a card with reaction circles. The tube agglutination test is a more sensitive and accurate method that can be done in glass tubes with serial dilutions of the serum. Both methods require observation for agglutination within a minute or after overnight incubation, depending on the type of antigen used.

The Widal test can detect both qualitative and quantitative aspects of the antigen-antibody reaction. Qualitative detection means determining the presence or absence of antibodies in the serum, while quantitative detection means measuring the amount or titer of antibodies in the serum. The titer is expressed as the reciprocal of the highest dilution of serum that still shows agglutination. For example, if agglutination occurs up to 1:80 dilution but not beyond, then the titer is 80. A titer greater than 1:80 is considered significant for diagnosis of enteric fever.

The Widal test was first introduced by Georges Fernand Isidor Widal in 1896 to diagnose Salmonella Paratyphi B infection. Later, it was modified and used to diagnose all types of enteric fever caused by Salmonella Typhi and Paratyphi. It is still widely used as a major diagnostic tool for enteric fever in many parts of the world.

To perform the Widal test, you will need the following materials and equipment:

• Blood sample of the suspected patient or serum
• Widal test kit containing Salmonella antigens
  • O-antigen of Salmonella Typhi
  • H-antigen of Salmonella Typhi
  • AH-antigen of Salmonella Paratyphi A
  • BH-antigen of Salmonella Paratyphi B
• Positive control
• Widal test card (glass slide can be used in substitution of card)
• Applicator stick
• Isotonic (0.85%) sterile saline solution
• Glass tubes
• Pipettes
• Incubator

The blood sample or serum should be fresh and stored at 2-8°C. The serum should not be heated or inactivated. The Widal test kit should contain standardized and stained antigens of Salmonella that are ready to use. The positive control should contain known antibodies against Salmonella antigens to verify the validity of the test. The Widal test card or slide should have separate reaction circles for each antigen and control. The applicator stick should be used to mix the serum and antigen solutions on the card or slide. The isotonic sterile saline solution should be used to dilute the serum for the tube agglutination test. The glass tubes should be clean and dry and labeled according to the antigen and dilution. The pipettes should be used to transfer the serum and antigen solutions accurately. The incubator should be set at 37°C for the tube agglutination test.

Widal test can be performed by two methods: rapid slide agglutination test and tube agglutination test. Slide agglutination test can be either qualitative or quantitative, while tube agglutination test is always quantitative. The slide agglutination test is faster and easier, but the tube agglutination test is more sensitive and accurate.

Rapid Slide Agglutination Test

This test is performed on a Widal test card or a glass slide with marked circles for each antigen and control. The test can be done in the following steps:

• Place a drop of sample serum in each reaction circle labeled as O, H, AH, and BH according to the antigens used for testing.
• Place a drop of positive control reagent in the reaction circle labeled as PC and a drop of negative control reagent in the reaction circle labeled as NC.
• Add one drop of each antigen solution (O, H, AH, and BH) over the corresponding serum and control circles.
• Mix the serum and antigen solutions uniformly using an applicator stick or a loop.
• Rock the slide gently to rotate the reaction mixture in a circular motion within the reaction circles.
• Observe for macroscopic agglutination (visible clumping) within a minute.

If agglutination occurs, the test is considered positive for that antigen. If no agglutination occurs, the test is considered negative.

Quantitative (Semi-quantitative) Slide Test

This test is performed to measure the level of antibodies present in the serum sample. This test is only performed if the qualitative test is positive. The test can be done in the following steps:

• Place a drop of normal saline into the first reaction circle.
• Place different volumes of the test sample (serum) in the remaining reaction circles, such as 5, 10, 20, 40, and 80 μl.
• Add a drop of the antigen solution that showed positive result in the qualitative test in each of the reaction circles.
• Mix the contents uniformly using an applicator stick or a loop.
• Rock the slide gently to rotate the reaction mixture in a circular motion within the reaction circles.
• Observe for macroscopic agglutination within a minute.

The antibody titer of the test serum is read as the highest serum dilution that gives a positive reaction. Antibody titer greater than 1:80 is considered significant.

Tube Agglutination Test (Quantitative Tube Test)

This test is performed in glass tubes with serial dilutions of the serum sample. This test detects the presence and level of antibodies against each antigen. The test can be done in the following steps:

• Arrange four sets of clean dry tubes (such as Kahn tubes) each set with eight tubes labeled as 1, 2, 3, 4, 5, 6, 7, and 8.
• Add 1.9 ml of isotonic (0.85%) sterile saline solution in tube 1 of each set.
• Add 1.0 ml of isotonic sterile saline in remaining tubes (2 – 8) of each set.
• Add 0.1 ml of sample serum in tube 1 of each set and mix properly.
• From tube 1 of each set, transfer 1.0 ml of diluted serum to tube 2 of their respective set.
• From tube 2 of each set, transfer 1.0 ml of diluted serum to tube 3 of their respective set.
• Continue this serial dilution process up to tube 7 of each set. From tube 7 of each set, discard 1.0 ml of dilution. (Never transfer to tube 8).
• Add one drop of Widal antigen-O in each tube of set 1, one drop of Widal antigen-H in each tube of set 2, one drop of Widal antigen-AH in each tube of set 3, and one drop of Widal antigen-BH in each tube of set 4.
• Mix the reaction mixture properly, cover the tubes and incubate at 37°C for about 18 hours (overnight).
• Following incubation look for agglutination at the bottom or along the sides of the tubes.

The antibody titer is read as the highest serum dilution that gives a positive reaction. Antibody titer greater than 1:80 is considered significant.

Widal antigens are the specific antigens of Salmonella Typhi and Paratyphi that are used to detect the presence of antibodies in the serum of patients with enteric fever. There are four types of Widal antigens: O, H, AH, and BH. O antigen is the somatic antigen of Salmonella Typhi, H antigen is the flagellar antigen of Salmonella Typhi, AH antigen is the flagellar antigen of Salmonella Paratyphi A, and BH antigen is the flagellar antigen of Salmonella Paratyphi B.

Widal antigens can be prepared in the laboratory using pure cultures of Salmonella Typhi and Paratyphi A and B, or they can be obtained from commercially available kits. The general steps for preparing Widal antigens are as follows :

• For preparing H antigens, an overnight broth culture or a saline suspension of an agar culture of Salmonella is treated with 0.1% formalin solution to kill and preserve the bacteria.
• For preparing O antigens, Salmonella is grown on phenol agar (1:800) to inhibit the formation of flagella. The growth is then emulsified in a small volume of saline, mixed with 20 times its volume of absolute ethanol, and heated at 40°C to 50°C for 30 minutes to extract the O antigen. The mixture is then centrifuged to separate the antigen from the bacterial debris.
• The antigens are treated with chloroform as a preservative and stained with suitable dyes for easy identification. For example, O antigen is stained with blue dye, H antigen with yellow dye, AH antigen with red dye, and BH antigen with green dye.
• The antigens are standardized by titrating them against known positive and negative sera to obtain optimal concentrations for agglutination reactions.

The prepared Widal antigens can be stored at 4°C for up to six months. They should be checked periodically for contamination, turbidity, or loss of reactivity. They should also be brought to room temperature before use.

This test is performed on the Widal test card. This is the most widely performed test and is of two types, viz;

Qualitative Slide Test

This test is done to detect the presence or absence of Salmonella antigens in the patient`s serum. It is a simple and quick method, but it cannot measure the antigen levels. It is performed in the following steps:

• Place a drop of sample serum in each reaction circle of the Widal test card labeled as O, H, AH, and BH.
• Place a drop of positive control reagent in the reaction circle labeled as PC
• Place a drop of negative control reagent in the reaction circle labeled as NC
• Add one drop of antigen solution H from the Widal test kit over the positive control and negative control
• Add a drop each of antigen solution O, H, AH, and BH over the sample serum in their respective reaction circle
• Mix the serum and antigen solution in each reaction circle uniformly using an applicator stick
• Rock the slide gently to rotate the reaction mixture in a circular motion within the reaction circles
• Observe for macroscopic agglutination within a minute.

Quantitative (Semi-quantitative) Slide Test

This test is performed to measure the level of Salmonella antigens present in the serum sample. This is only performed if the qualitative test is positive. It is performed in the following steps:

• Place a drop of normal saline into the first reaction circle
• Place 5, 10, 20, 40, and 80 μl of the test sample (serum) in the remaining reaction circles respectively
• Add a drop of antigen suspension that showed positive test (i.e. agglutination) in the qualitative test in each of the reaction circles
• Mix the contents uniformly using an applicator stick
• Rock the slide gently to rotate the reaction mixture in a circular motion within the reaction circles
• Observe for macroscopic agglutination within a minute.

The antibody titer of the test serum is read as the highest serum dilution that gives a positive reaction (visible agglutination). Antibody titer greater than 1:80 is considered significant .

This test is performed in glass tubes and is more sensitive and accurate than the slide agglutination test. It detects the presence of antibodies as well as measures the antibody titer. The test is performed in the following steps:

• Arrange 4 sets of clean dry Khan tubes, each set with 8 tubes labeled as 1, 2, 3, 4, 5, 6, 7, and 8.
• Add 1.9 ml of isotonic (0.85%) sterile saline solution in tube 1 of each set.
• Add 1.0 ml of isotonic sterile saline in remaining tubes (2 – 8) of each set.
• Add 0.1 ml of sample serum in tube 1 of each set and mix properly.
• From tube 1 of each set, transfer 1.0 ml of diluted serum to each tube 2 of their respective set.
• From tube 2 of each set, transfer 1.0 ml of diluted serum to each tube 3 of their respective set.
• Continue this serial dilution process up to tube 7 of each set. From tube 7 of each set, discard 1.0 ml of dilution. (Never transfer to tube 8).
• Add 1 drop of Widal antigen-O in each tube of set 1, 1 drop of Widal antigen-H in each tube of set 2, 1 drop of Widal antigen-AH in each tube of set 3, and 1 drop of Widal antigen-BH in each tube of set 4.
• Mix the reaction mixture properly, cover the tubes and incubate at 37°C for about 18 hours (overnight).
• Following incubation look for agglutination at the bottom of the tubes.

The antibody titer is read as the highest serum dilution that gives a positive reaction (visible agglutination). Antibody titer greater than 1:80 is considered significant . The titer can be read according to the table below:

A positive result is indicated by a clump formation at the bottom of the tube and a negative result is indicated by a clear suspension .

The interpretation of Widal test results depends on the method used, the type of antigen, and the level of antibody titer. The antibody titer is the highest dilution of serum that shows a positive agglutination reaction with the antigen.

Qualitative Slide Test Results

• Positive test (presence of Salmonella antibodies) = Agglutination within a minute
• Negative test (absence of Salmonella antibodies) = No agglutination

Quantitative Slide Test Results

• The antibody titer of test serum is read as the highest serum dilution that gives a positive reaction (visible agglutination).
• Antibody titer greater than 1:80 is considered significant and usually suggests Salmonella infection .
• A single positive test is less significant than a rising antibody titer, since a rising titer is considered to be a definite evidence of infection.
• The titer can be read according to the table below:

Quantitative Tube Test Results

• The antibody titer is read as the highest serum dilution that gives a positive reaction (visible agglutination).
• Antibody titer greater than 1:80 is considered significant and usually suggests Salmonella infection .
• A single positive test is less significant than a rising antibody titer, since a rising titer is considered to be a definite evidence of infection.
• The titer can be read according to the table below:

Factors affecting Widal Test Results

• The interpretation of Widal test results should take into account the following factors:

  • The endemicity of Salmonella infection in the region
  • The previous exposure or vaccination of the patient
  • The time of specimen collection after the onset of symptoms
  • The presence of other infections or conditions that may cause cross-reactivity
  • The sensitivity and specificity of the antigen and antibody reagents
  • The quality control and standardization of the test procedure
  • Applications of Widal Test
  
  

• The Widal test is used for the rapid diagnosis of enteric fever (typhoid and paratyphoid fever) in endemic areas where culture facilities are not available or reliable.

• The Widal test can identify the serovar (Typhi, Paratyphi A, B or C) of Salmonella causing enteric fever by detecting the specific antibodies against O and H antigens of the bacterium.
• The Widal test can also help to monitor the response to treatment and the development of immunity in patients with enteric fever by measuring the changes in antibody titers over time.

Limitations of Widal Test

Widal test is a widely used serological test for the diagnosis of enteric fever, but it has some limitations that affect its accuracy and reliability. Some of the limitations are:

• Widal test is time-consuming to find antibody titre and often times when the diagnosis is reached it is too late to start an antibiotic regimen .
• Widal test may be falsely positive in patients who have had previous vaccination or infection with S. Typhi . It may also cross-react with antibodies produced against other Salmonella species, typhus, malaria, and other infections.
• Widal test is dependent on the local titre of the place where the test is performed. The local titre may vary depending on the endemicity of enteric fever, the prevalence of chronic carriers, and the vaccination status of the population . Therefore, a single titre value may not be universally applicable for all regions and populations.
• Widal test requires a paired sera sample taken at an interval of 7 to 10 days to demonstrate a rising antibody titre. A single serum sample may not reflect the true antibody level and may give false-negative results .
• Widal test is affected by the use of antibiotics and the immune status of the patient. Antibiotics may reduce the antibody response and lead to false-negative results. Similarly, immunocompromised patients may have a low level of antibody production and give false-negative results .
• Widal test has low sensitivity and specificity compared to other diagnostic methods such as blood culture, stool culture, and molecular techniques. It may miss some cases of enteric fever or give false-positive results for other infections .

Due to these limitations, Widal test is considered a presumptive test and should be interpreted with caution and clinical correlation. It should not be used as the sole criterion for the diagnosis of enteric fever. It should be supplemented by other laboratory tests and epidemiological data to confirm the diagnosis .

Widal test is a widely used serological test for the diagnosis of enteric fever, but it has some limitations that affect its accuracy and reliability. Some of the limitations are:

• Widal test is time-consuming to find antibody titre and often times when the diagnosis is reached it is too late to start an antibiotic regimen .
• Widal test may be falsely positive in patients who have had previous vaccination or infection with S. Typhi . It may also cross-react with antibodies produced against other Salmonella species, typhus, malaria, and other infections.
• Widal test is dependent on the local titre of the place where the test is performed. The local titre may vary depending on the endemicity of enteric fever, the prevalence of chronic carriers, and the vaccination status of the population . Therefore, a single titre value may not be universally applicable for all regions and populations.
• Widal test requires a paired sera sample taken at an interval of 7 to 10 days to demonstrate a rising antibody titre. A single serum sample may not reflect the true antibody level and may give false-negative results .
• Widal test is affected by the use of antibiotics and the immune status of the patient. Antibiotics may reduce the antibody response and lead to false-negative results. Similarly, immunocompromised patients may have a low level of antibody production and give false-negative results .
• Widal test has low sensitivity and specificity compared to other diagnostic methods such as blood culture, stool culture, and molecular techniques. It may miss some cases of enteric fever or give false-positive results for other infections .

Due to these limitations, Widal test is considered a presumptive test and should be interpreted with caution and clinical correlation. It should not be used as the sole criterion for the diagnosis of enteric fever. It should be supplemented by other laboratory tests and epidemiological data to confirm the diagnosis .

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