Widal Test – Principle, Procedure, Result, Advantages, Limitation

What is Widal Test?

• The Widal test is a serological agglutination test that is commonly used in clinical laboratories for the diagnosis of enteric fever, specifically Typhoidal or Paratyphoidal fever. This test measures the level of agglutinating serum antibodies produced against the O (somatic) and H (flagellar) antigens of Salmonella Typhi and Paratyphi A, B, and C.
• The principle behind the Widal test is based on the antigen-antibody agglutination reaction. Commercially available purified antigens of the Salmonella strains causing enteric fevers are used in the test. The patient’s serum is tested against these antigens, and if visible agglutination occurs, the test is considered positive.
• Salmonella Typhi has O and H antigens on its cell wall and flagella, respectively. Salmonella Paratyphi A and Paratyphi B have AH and BH antigens on their flagella, respectively. The O antigen of Salmonella Paratyphi is not used in the test because it cross-reacts with the O antigens of Salmonella Typhi.
• When the antigens are introduced into the host’s immune system, they induce the production of specific antibodies. These antibodies become detectable in the serum after 6-8 days of infection. The Widal test detects these specific antibodies through an agglutination reaction.
• The Widal test was first introduced in 1896 by Georges Fernand Isidor Widal, a French physician and bacteriologist. Initially, the test was used to diagnose Salmonella Paratyphi B infection, but it was later modified and utilized for diagnosing all types of enteric fevers.
• The Widal test is an agglutination testing method that detects antibodies in the patient’s serum, which are produced in response to the causative agents of typhoid and paratyphoid illnesses, namely Salmonella Typhi and Salmonella Paratyphi A, B, and C. It is widely used, particularly in endemic areas, as a primary tool for identifying the causative agents of these febrile illnesses.
• The test utilizes the interaction between antigens and antibodies to determine the specific antibodies present in the patient’s serum. The antigens used in the test are derived from the bacteria that cause Salmonella Typhi and Salmonella Paratyphi infections.
• Salmonella Typhi has O antigen on its cell wall and H antigens on its flagella, while Salmonella Paratyphi A and Paratyphi B have O antigens on their cell walls and AH and BH antigens on their flagella, respectively.
• During an infection, these antigens stimulate the body to produce specific antibodies, which are released into the bloodstream. These specific antibodies can be detected in the patient’s serum within the first six days after the onset of the fever.
• In summary, the Widal test is a widely used agglutination test for diagnosing enteric fever caused by Salmonella Typhi and Salmonella Paratyphi A, B, and C. It relies on the detection of specific antibodies produced in response to these bacteria, using commercially available antigens.

Definition of Widal Test

The Widal test is a serological agglutination test used to diagnose enteric fever caused by Salmonella Typhi and Salmonella Paratyphi. It detects specific antibodies in the patient’s serum that are produced in response to these bacteria.

Objective of Widal Test

The main objective of the Widal test is to detect the presence of specific serum antibodies against Salmonella Typhi and Paratyphi. By measuring the agglutinating serum antibodies, the test aims to diagnose enteric fever caused by these bacteria.

The presence of antibodies indicates that the patient has been exposed to or is currently infected with Salmonella Typhi or Paratyphi. The test helps in the diagnosis and differentiation of enteric fever from other febrile illnesses.

The Widal test is particularly useful in areas where enteric fever is endemic and serves as a major diagnostic tool for identifying the causative agent. The test results aid healthcare professionals in making accurate diagnoses, initiating appropriate treatment, and implementing necessary preventive measures.

Overall, the objective of the Widal test is to detect specific antibodies against Salmonella Typhi and Paratyphi in order to diagnose enteric fever and guide appropriate patient management.

Widal Test Principle

The Widal test is based on the principle of antigen-antibody reaction of the agglutination type. The patient’s serum is mixed with a suspension of killed bacteria, specifically Salmonella carrying O, H, AH, and BH antigens. If the patient has homologous antibodies against these antigens, a visible clumping reaction (agglutination) occurs.

The antibodies against O and H antigens of Salmonella Typhi and Paratyphi are produced in high levels in the serum of patients infected with these bacteria during enteric fever. After 6-8 days of infection, the antibody levels peak. The patient’s serum is collected and mixed with the specifically prepared O and H antigens of Salmonella Typhi, as well as AH and BH antigens of Salmonella Paratyphi. If antibodies are present in the serum, they will agglutinate the corresponding antigens, leading to visible clumping.

When someone consumes food contaminated with Salmonella bacteria, the antigens from the bacteria enter their body. The immune system then produces antibodies against these antigens. In the Widal test, the antibodies present in the patient’s serum react with the insoluble O and H antigens of Salmonella Typhi and Paratyphi, causing visible clumping (agglutination) to occur.

This agglutination test detects the presence of antibodies that the body has produced in response to a specific bacteria or antigen. In the case of typhoid fever, the patient’s serum will contain antibodies that react and agglutinate the Salmonella antigens in the Widal test.

In summary, the principle of the Widal test is that if specific antibodies against the O and H antigens of Salmonella Typhi and Paratyphi are present in the serum, they will react with the corresponding antigens and produce visible clumping, indicating a positive test result.

Requirements for Widal Test

1. Blood sample or serum: A fresh serum sample or blood sample from the suspected patient is needed for testing.
2. Widal test kit: The kit should contain vials with stained Salmonella antigens. Specifically, it should include the following antigens:
   • O antigen of Salmonella Typhi
   • H antigen of Salmonella Typhi
   • AH antigen of Salmonella Paratyphi A
   • BH antigen of Salmonella Paratyphi B
3. Positive control: A positive control sample is included in the kit to validate the test and ensure accurate results.
4. Widal test card or slide: A Widal test card, which is specifically designed for the test, is commonly used. However, a glass slide can also be used as a substitute.
5. Applicator stick: An applicator stick is required for mixing and transferring the serum and antigens during the test.

It is important to note that the serum should be fresh and stored at a temperature between 2°C and 8°C. Heating or inactivation of the serum should be avoided to maintain the integrity of the antibodies.

These are the basic requirements for conducting the Widal test, and they are essential for performing the test accurately and obtaining reliable results.

Widal Test Procedures

Widal testing is performed using two methods: rapid slide agglutination and tube agglutination. Agglutination on slides can serve as either a qualitative or quantitative test. The tube method is more sensitive and accurate than the slide agglutination method, but the slide method is more popular.

Preparation of Widal Antigens

1. H suspension preparation: A 24-hour broth culture or saline suspension of an agar culture is prepared. To this culture, 0.1% formalin is added. Formalin acts as a fixative and helps in preserving the antigenic properties of the bacteria. The formalin-treated culture is incubated for a specific period.
2. O suspension preparation: The bacteria are cultured on phenol agar at a dilution of 1:800. The phenol present in the agar inhibits the formation of flagella, which are the components responsible for the O antigens. This step ensures that the O antigens are not present in the final suspension.
3. Selection of standard smooth strains: Standard smooth strains of the target organism are used for the preparation of Widal antigens. In the case of Widal testing, the strains commonly employed are S. Typhi 901. These strains are chosen for their well-characterized antigenic properties.
4. Emulsification and purification: The growth from the selected strains is emulsified in a small volume of saline. The bacterial suspension is then mixed with 20 times its volume of alcohol, typically ethanol. This mixture is heated at a temperature between 40°C to 50°C for 30 minutes. The heat treatment kills the bacterial cells while preserving the antigens. After heating, the mixture is centrifuged to separate the bacterial debris and other impurities from the antigen solution.
5. Treatment with chloroform and staining: To ensure the stability and longevity of the antigens, a preservative such as chloroform is added. Chloroform helps in preventing the growth of microorganisms and preserving the antigenic properties of the solution. Finally, appropriate dyes are added to the antigen suspension for easy identification during diagnostic tests. These dyes aid in distinguishing between different antigens and enhance visibility.

These steps are followed to prepare Widal antigens in the laboratory. The prepared antigens can be used in the Widal test, a serological diagnostic test for detecting antibodies against Salmonella Typhi and Paratyphi A and B in patient serum samples.

Rapid Slide Agglutination Test

The Widal test card is used for this test. This is the most commonly used test and comes in two varieties:

A. Qualitative Slide Test

The Qualitative Slide Test is a diagnostic method used to determine the presence or absence of Salmonella antigens associated with enteric fever. This test is simple and provides quick results, although it does not allow for the measurement of antigen levels. The test is performed following these steps:

1. Widal Test Card: The Widal test card contains reaction circles labeled as O, H, AH, BH, PC (positive control), and NC (negative control).
2. Sample Serum: A drop of the patient’s serum sample is placed in each reaction circle labeled as O, H, AH, and BH. These reaction circles correspond to specific Salmonella antigens.
3. Positive and Negative Controls: A drop of the positive control reagent is placed in the reaction circle labeled as PC, while a drop of the negative control reagent is placed in the reaction circle labeled as NC. These controls serve as reference points for interpreting the test results.
4. Antigen Solution: One drop of the antigen solution H, obtained from the Widal test kit, is added over the positive control and negative control reaction circles.
5. Antigen Solution and Serum: A drop each of the antigen solution O, H, AH, and BH is added over the sample serum in their respective reaction circles. These antigen solutions contain specific Salmonella antigens.
6. Mixing the Reaction Mixture: Using an applicator stick, the serum and antigen solutions in each reaction circle are mixed uniformly. This ensures proper contact between the antigens and the patient’s serum.
7. Circular Motion: The slide is gently rocked or rotated in a circular motion to allow the reaction mixture to move within the reaction circles. This motion aids in the detection of any macroscopic agglutination, which indicates the presence of antibodies against the Salmonella antigens.
8. Observation: The test results are observed within a minute. The presence of macroscopic agglutination, which appears as clumping or aggregation of particles, indicates a positive result for the corresponding antigen in the patient’s serum.

The Qualitative Slide Test is a valuable screening tool for detecting Salmonella antigens in cases of enteric fever. However, it is important to note that this test provides a qualitative result (positive or negative) and does not quantify the antigen levels in the serum. Further confirmatory testing may be necessary to determine the specific antibody titers or to obtain quantitative results.

2. Quantitative (Semi-quantitative) Slide Test

The Quantitative (Semi-quantitative) Slide Test is a diagnostic method used to measure the level of antigens present in a serum sample. This test is performed when the qualitative test, which detects the presence or absence of antigens, yields a positive result. The steps involved in performing the test are as follows:

1. Normal Saline: A drop of normal saline is placed into the first reaction circle on the slide. This serves as a control and helps create a baseline for comparison.
2. Test Sample: In the remaining reaction circles on the slide, 5, 10, 20, 40, and 80 μl of the serum sample (test sample) are placed, each in a separate reaction circle. These different volumes of the serum sample allow for a semi-quantitative assessment of the antigen levels.
3. Positive Antigen Suspension: A drop of the antigen suspension that showed a positive result (agglutination) in the qualitative test is added to each of the reaction circles containing the test samples. This positive antigen suspension contains the specific antigens of interest.
4. Mixing the Reaction Mixture: Using an applicator stick, the contents of each reaction circle are mixed uniformly. This ensures proper contact between the antigen suspension and the test samples.
5. Circular Motion: The slide is gently rocked or rotated in a circular motion to allow the reaction mixture to move within the reaction circles. This motion facilitates the detection of any macroscopic agglutination, indicating the presence of antigen-antibody interactions.
6. Observation: The test results are observed within a minute. The presence of macroscopic agglutination, visible as clumping or aggregation of particles, indicates a positive result. The degree of agglutination is then visually compared across the reaction circles containing different volumes of the test sample. This provides a semi-quantitative assessment of the antigen levels present in the serum sample.

The Quantitative (Semi-quantitative) Slide Test allows for a rough estimation of the antigen levels in a serum sample. However, it is important to note that this method provides a relative measurement and may not yield precise quantitative results. For more accurate and precise quantification of antigen levels, alternative methods such as enzyme-linked immunosorbent assays (ELISA) or other specialized laboratory techniques may be necessary.

Tube Agglutination Test (Quantitative Tube Test)

The Tube Agglutination Test, also known as the Quantitative Tube Test, is a serological method used to detect the presence of antibodies and measure their titer in a serum sample. This test is performed using glass tubes, and although different types of tubes were originally used, modern methods often employ Khan tubes or simple test tubes. The test is conducted as follows:

1. Preparation of Tube Sets: Arrange four sets of clean and dry Khan Tubes, with each set containing eight tubes. Label the tubes in each set as 1, 2, 3, 4, 5, 6, 7, and 8.
2. Saline Solution: In tube 1 of each set, add 1.9 ml of isotonic (0.85%) sterile saline solution. In the remaining tubes (2 to 8) of each set, add 1.0 ml of isotonic sterile saline solution.
3. Sample Serum: Add 0.1 ml of the serum sample to tube 1 of each set, and mix it thoroughly with the saline solution.
4. Serial Dilution: From tube 1 of each set, transfer 1.0 ml of the diluted serum to tube 2 of the respective set. Repeat this serial dilution process, transferring 1.0 ml of diluted serum from each preceding tube to the next tube in the set, up to tube 7. In tube 7 of each set, discard 1.0 ml of the dilution (do not transfer it to tube 8).
5. Antigen Addition: Add 1 drop of Widal antigen-O to each tube of set 1, 1 drop of Widal antigen-H to each tube of set 2, 1 drop of Widal antigen-AH to each tube of set 3, and 1 drop of Widal antigen-BH to each tube of set 4. These antigens correspond to specific Salmonella antigens.
6. Mixing and Incubation: Mix the contents of each tube properly, cover the tubes, and incubate them at 37°C for approximately 18 hours (overnight). This allows time for the antigen-antibody reactions to occur.
7. Agglutination Observation: After the incubation period, examine the tubes for agglutination. Agglutination appears as clumping or agglutinated particles settled at the bottom of the tubes.

By observing the presence or absence of agglutination in the tubes, the Tube Agglutination Test can provide information about the presence of antibodies and their titer in the serum sample. The higher the dilution at which agglutination is observed, the higher the antibody titer against the specific Salmonella antigens being tested. This test is valuable in diagnosing certain infections, including enteric fevers caused by Salmonella strains, and monitoring the immune response of individuals to these infections.

Result Interpretation of Widal Test

Qualitative Slide Test Results

The Qualitative Slide Test is a diagnostic method used to determine the presence or absence of Salmonella antigens. The test results are interpreted based on the occurrence or absence of agglutination within a minute of performing the test. The interpretations are as follows:

• Positive Test (Presence of Salmonella Antigen): If agglutination, which appears as clumping or aggregation of particles, is observed within a minute after combining the antigen solution and the patient’s serum in the reaction circles, the test is considered positive. This indicates the presence of Salmonella antigens in the patient’s serum, suggesting a possible infection caused by Salmonella.
• Negative Test (Absence of Salmonella Antigen): If no agglutination is observed within a minute, the test is considered negative. This means that there are no detectable Salmonella antigens in the patient’s serum, suggesting the absence of a current infection caused by Salmonella.

The Qualitative Slide Test results provide a qualitative indication of the presence or absence of Salmonella antigens in the patient’s serum. It is important to note that this test does not provide quantitative information regarding the level or concentration of the antigens. For a more detailed assessment or to measure the antibody titer, additional tests such as the Quantitative Slide Test or Tube Agglutination Test may be performed.

Quantitative (Semi-quantitative) Slide Test Results

The Quantitative (Semi-quantitative) Slide Test is a diagnostic method used to measure the antibody titer in a serum sample. The antibody titer is determined by identifying the highest serum dilution that results in a positive reaction, which is indicated by visible agglutination. The test results are interpreted based on the dilution and corresponding antibody titer. The following table illustrates the interpretation of the results:

Reaction Circle	Volume of Serum	Antibody Titer
2nd	5μl	1:320
3rd	10 μl	1:160
4th	20 μl	1:80
5th	40 μl	1:40
6th	80 μl	1:20

For example, if agglutination is observed at the 4th reaction circle where 20 μl of the serum sample is used, the corresponding antibody titer is reported as 1:80. This indicates that the serum sample can be diluted up to 1:80 and still show visible agglutination.

A significant antibody titer is generally considered to be greater than 1:80. This means that a dilution of 1:80 or higher is required to observe agglutination. Antibody titers can vary depending on the specific disease or condition being tested, so the significance of the titer may vary in different clinical contexts.

The Quantitative (Semi-quantitative) Slide Test provides a relative measurement of the antibody titer, allowing for a rough estimation of the antibody levels in the serum sample. It is important to note that this method does not provide precise quantitative results. For a more accurate and precise quantification of antibody titers, further testing with quantitative methods, such as the Tube Agglutination Test or enzyme-linked immunosorbent assays (ELISA), may be necessary.

Tube Agglutination Test (Quantitative Tube Test) Results

The Tube Agglutination Test, also known as the Quantitative Tube Test, is a serological method used to measure the antibody titer in a serum sample. The antibody titer is determined by identifying the highest serum dilution that results in a positive reaction, which is indicated by visible agglutination. The test results are interpreted based on the dilution and corresponding antibody titer. The following table illustrates the interpretation of the results:

Tube Number	Antibody Titer
1	1:20
2	1:40
3	1:80
4	1:160
5	1:320
6	1:640
7	1:1280
8	Negative control

For example, if agglutination is observed up to the 5th tube, which has a dilution of 1:320, the corresponding antibody titer is reported as 1:320. This means that a dilution of 1:320 or higher is required to observe agglutination.

A significant antibody titer is generally considered to be greater than 1:80. This means that a dilution of 1:80 or higher is required to observe agglutination. Antibody titers can vary depending on the specific disease or condition being tested, so the significance of the titer may vary in different clinical contexts.

It is important to note that the 8th tube serves as a negative control. It does not contain any serum sample and is used as a reference to confirm that no agglutination occurs without the presence of antibodies.

The Tube Agglutination Test provides a quantitative measurement of the antibody titer, allowing for a more precise assessment of the antibody levels in the serum sample compared to qualitative tests. However, it is still a relatively rough estimation of the antibody titer and may not provide precise quantitative results. For more accurate and precise quantification of antibody titers, additional laboratory methods, such as enzyme-linked immunosorbent assays (ELISA) or other specialized techniques, may be necessary.

Widal Test Applications

The Widal Test is a serological method that has various applications in the diagnosis and identification of enteric fever caused by Salmonella bacteria. The following are some of the key applications of the Widal Test:

1. Rapid Diagnosis of Enteric Fever: The Widal Test is primarily used as a rapid diagnostic tool for enteric fever, which includes typhoid fever and paratyphoid fever. By detecting specific antibodies in the patient’s serum, the test helps in confirming the presence of Salmonella infection and aids in the diagnosis of enteric fever.
2. Identification of Serovar: The Widal Test is also utilized to identify the serovar or specific strain of Salmonella causing enteric fever. It can differentiate between different serovars such as Salmonella Typhi, Paratyphi A, and Paratyphi B. This information is valuable for appropriate treatment and management of the infection.
3. Rapid Screening in Endemic Areas: In endemic areas where enteric fever is prevalent, the Widal Test serves as a rapid screening tool for detecting cases of typhoid fever. It allows for quick identification of individuals who may require further diagnostic evaluation or treatment, helping in the early management of the disease.
4. Reliable Alternative to Culture Facilities: In situations where culture facilities are not readily available or where obtaining a bacterial culture is challenging, the Widal Test is considered a reliable alternative for diagnosing enteric fever. It provides valuable diagnostic information without the need for specialized laboratory equipment or techniques.
5. Detection of Salmonella Typhi and Paratyphi: The Widal Test is applicable for both Salmonella Typhi and Salmonella Paratyphi, which are the main causative agents of enteric fever. It helps in detecting antibodies specific to these bacteria, aiding in the diagnosis and differentiation of typhoid and paratyphoid fever.

Overall, the Widal Test has wide-ranging applications in the diagnosis, identification, and screening of enteric fever caused by Salmonella bacteria, particularly in settings where rapid diagnosis is crucial or when culture facilities are limited. However, it is important to note that the test has certain limitations and should be interpreted in conjunction with clinical symptoms, patient history, and other diagnostic methods for a comprehensive assessment of the disease.

Advantages of Widal Test

The Widal Test offers several advantages in the diagnosis and identification of enteric fever caused by Salmonella bacteria. Here are some of its key advantages:

1. Fast Test: The Widal Test provides a rapid method for determining the presence of enteric fever, particularly in regions with a high incidence of the disease. This allows for quick diagnosis and timely initiation of treatment, leading to better patient management.
2. Accessibility: In areas where culture facilities may not be readily accessible, the Widal Test is a valuable tool. It offers a reliable alternative to bacterial culture, enabling healthcare providers to make informed diagnostic decisions even in resource-limited settings.
3. Identification of Salmonella Strains: The Widal Test is capable of identifying diseases caused by both Salmonella Typhi and Salmonella Paratyphi. This is important as these serovars are responsible for the majority of cases of enteric fever. The test helps differentiate between different strains, aiding in appropriate treatment and management.
4. Cost-Effective: Compared to more advanced laboratory techniques, the Widal Test is relatively cost-effective. It requires minimal equipment and resources, making it accessible in healthcare facilities with limited budgets.
5. Wide Availability: The Widal Test is widely available and routinely used in many regions where enteric fever is prevalent. It is a well-established serological test that has been in use for several decades, and its availability contributes to its usefulness in clinical practice.

While the Widal Test offers these advantages, it is important to note that it also has limitations. False-positive and false-negative results can occur, and interpretation of results must be done cautiously, considering clinical symptoms, patient history, and other diagnostic tests for a comprehensive assessment of the disease.

Limitations of Widal Test

The Widal Test, despite its usefulness in diagnosing enteric fever caused by Salmonella bacteria, has several limitations that must be considered. These limitations include:

• False-Positive Results: Vaccinated or previously infected individuals may produce false-positive results, leading to inaccurate interpretations of the test.
• Timing of Agglutinin Appearance: Agglutinins, the antibodies detected in the Widal Test, typically appear in the serum 6-8 days after infection and persist for up to 4 weeks. Consequently, the test has low sensitivity and efficiency when performed before the first week or after the fourth week of infection.
• Presumptive Nature: The Widal Test is considered a presumptive test and requires further confirmatory investigations to establish an enteric fever diagnosis. Reliance solely on the Widal Test may result in misdiagnosis.
• Cross-Reactivity: The test may exhibit cross-reactivity with antibodies produced against other diseases, such as typhus, malarial parasites, and non-enteric Salmonella infections. This cross-reactivity can lead to false-positive results.
• Time Requirement for Quantitative Tube Tests: The quantitative tube tests, used to determine antibody titers, require a longer time for incubation and observation compared to other rapid diagnostic methods.
• Antibiotic Treatment and Immune Disorders: Antibiotic treatment can reduce antibody responses, potentially leading to false-negative results. Similarly, individuals with immune disorders may exhibit low levels of antibody production, further contributing to false-negative outcomes.
• Variations in Clinical Significance: The clinical significance of agglutinin titers may differ between different geographical locations and individuals, making it challenging to establish universally applicable cutoff levels for interpretation.
• Time Consumption: Determining antibody titers through the Widal Test can be time-consuming. In some cases, by the time the diagnosis is confirmed, it may already be too late to initiate an antibiotic regimen.
• Inability to Differentiate Current and Previous Infections: The Widal Test cannot differentiate between a current infection and a previous infection or vaccination against typhoid. This can lead to difficulties in determining the patient’s actual disease status.
• Cross-Reactivity with Other Salmonella Species: The test may show cross-reactivity with antibodies produced against other Salmonella species, potentially resulting in false-positive results.
• Influences on Baseline Titers: The interpretation of Widal test results should consider baseline titers in the local healthy population. However, these baseline antibody levels can vary over time and in different areas, making it challenging to establish consistent cutoff levels for accurate interpretation.
• Interference Factors: Factors such as severe hypoproteinaemia, early antimicrobial treatment, or typhoid relapse can interfere with antibody responses, leading to false-negative results.
• Variability Between Laboratories: Variations in the performance and reading of Widal tests between different laboratories can compromise the reliability and consistency of the test results.

Due to these limitations, the World Health Organization (WHO) recommends caution in relying solely on the Widal Test and suggests considering other diagnostic methods and clinical parameters to enhance accuracy in diagnosing enteric fever.

FAQ

References

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