Widal Agglutination Test: A rapid serological diagnosis of typhoid fever

Widal Agglutination Test: A rapid serological diagnosis of typhoid fever including slide qualitative test, Slide quantitative test , tube test method

1. NIKKI YADAV M.Sc. Biotechnology Widal Agglutination Test: A Rapid Serological Diagnosis of Typhoid Fever

2. OVERVIEW INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LIMITATIONS CONCLUSION

3. INTRODUCTION The Widal test, named after French bacteriologist Fernand Widal in 1896, is a diagnostic test used to detect the presence of antibodies against Salmonella typhi , the causative agent of typhoid fever. The Widal test is a serological diagnostic tool used to aid in the diagnosis of enteric fevers, particularly typhoid fever caused by Salmonella typhi and paratyphoid fever caused by Salmonella paratyphi A and B. This test measures the presence and levels of specific antibodies in a patient's serum against the O (somatic) and H (flagellar) antigens of these Salmonella species. Salmonella typhi and Salmonella paratyphi are the two types of bacteria. S. typhi has two antigens: S. typhi O (primary antigen) and S. typhi H (secondary antigen), S. paratyphi has the also have two antigens: S. paratyphi A and S. paratyphi B. Salmonella typhi and Salmonella paratyphi are two closely related bacteria that cause typhoid fever and paratyphoid fever, respectively.

4. O and H antigens of Salmonella typhi are the main reasons of causing disease and results of Widal test based on these antigens. Difference between O and H antigen:

5. Typhoid fever is a serious and potentially life-threatening bacterial illness caused by the bacteria Salmonella enterica serotype Typhi. Spread through the ingestion of contaminated food or water and is more prevalent in areas with poor sanitation and limited access to clean drinking water. Some signs and symptoms of typhoid fever which indicate you might be infected with the condition include: Irregular fever that can go as high as 104.9 ˚F (40.5 ˚C) Headache Pain in the abdomen Feeling of tiredness and weakness Muscle pain Loss of appetite/anorexia Nausea and vomiting Constipation or diarrhea Widal test has been in use for over a century and remains a widely employed diagnostic method, particularly in resource-limited settings where more advanced diagnostic techniques may not be readily available or affordable. However, it is important to note that the Widal test has limitations in terms of sensitivity and specificity, its interpretation requires careful consideration of clinical presentation and epidemiological factors.

6. MATERIALS AND METHODS The materials used in the Widal agglutination test typically include: Bacterial Antigens : O,H,AH,BH-antigens these antigens are often commercially available or prepared in specialized laboratories. Blood Sample: A small sample of the patient's blood is collected, and the serum is separated for testing. Saline Solution: A physiological saline solution (0.9% NaCl) is used to suspend the bacterial antigens and prepare dilutions of the patient's serum. Glass Slides Or Micro-titer Plates: These are used as a surface for mixing the serum and antigens, allowing for observation of agglutination reactions. Positive And Negative Controls: To ensure the validity and accuracy of the test. So before using kit we should follow precautions and follow some reagent storage and stability rules : Bring all the reagents and samples to room temperature before use. Shake all the antigens thoroughly before use. Store the reagent at 2-8°C, don’t`freeze. The reagent is light sensitive.The shelf life of the reagent is as per the expiry date mentioned on the reagent vial labels. Discard the reagent if they become contaminated or do not demonstrate correct activity with controls. Do not interchange the reagents from other batches. (The blue stained antigens are specific to the somatic antigens (O), while the red stained antigens are specific to the flagella antigen (H).

7. SLIDE WIDAL TEST: Slide Qualitative Widal Test: METHODS: For this test, you will use a slide with 6 reaction circles as shown in the figure, marked as O, H, AH,BH, PC and NC. To begin with; Put one drop of the patient’s serum in four reaction circles, i.e., O, H, AH, BH. Add one drop of positive control in the PC circle and one in the NC circle. 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. Add any antigen, i.e., O, H, AH, BH in both PC and NC. 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. Also, one mixture should not mix with another, as it can influence the test results. 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

8. Slide 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, we need to follow these steps: Use a different slide with 8 circles as shown in the fig.6: four for O antigen and four for H. 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. In the same way, put one drop of the specific reagent in all four circles. 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 (table). Mix the contents uniformly using applicator stick. Rock the slide gently to rotate the reaction mixture in circular motion within reaction circles. Observe for macroscopic agglutination within a minute. Titer Values For Slide Quantitative Method

9. TUBE Widal TEST METHOD: Apart from qualitative and quantitative Widal tests, there is another test method; the standard tube method. 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. To begin with; 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 ml 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). Titer Values of Tube Widal Test

10. RESULTS & DISCUSSION Slide Qualitative Test (Widal Test): Positive Result: If agglutination is observed in a well, it indicates the presence of antibodies against the corresponding Salmonella antigen. Slide Quantitative Test (Widal Test): Agglutination is a positive result indicating the presence of antibodies against the specific Salmonella antigens being tested. The interpretation of the slide quantitative Widal test results depends on the specific antibodies being tested (O antigen and H antigen for Salmonella typhi and paratyphi). For the diagnosis of a Widal blood test, titers need to be included in the diagnosis to obtain the typhoid test report. The Widal test normal range is when the titer is equal to or below 1:80 in both O and H antigens. Positive Result - A titer of O and H antigens greater than 1:60 to 1:320 is considered positive. Negative Result - A negative, commonly considered a normal range for a Widal test result is when the value of O and H antigens are less than 1:160. Salmonella typhi O (+ve) mean recent infection Salmonella typhi H (-ve) mean old infection Salmonella paratyphi BH (+ve) mean carrier can infect other. You have no typhoid as per the report and BH means that a paratyphi B has infected you any time in the past not now.

11. Results And Titer Values of a Slide Widal Test to Identify infection Agglutination Reaction Of Antigens (O,H,AH,BH) Highly O+ve & Mild H+ve AH+ve No Agglutination (Negative Result) Tube Widal Test: 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 titer 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.

12. LIMITATIONS & ALTERNATIVES OF WIDAL TEST Cross-Reactivity With Other Infections. Timing of Antibody Production. Variability In Antibody Levels. Limited Specificity. Interpretation Challenges. Need For Confirmatory Tests. False-Positive Results In Vaccinated Individuals. Inability To Differentiate Between Current And Past Infections. Reduced Sensitivity. LIMITATIONS Rapid Diagnostic Tests (RDTs). TPTest. Blood Culture. Immunochromatographic Tests (ICTs). ELISA PCR Tubex Typhidot ALTERNATIVES OF WIDAL TEST

13. CONCLUSION The Widal test is a valuable diagnostic tool for detecting previous exposure to Salmonella typhi, the causative agent of typhoid fever. By measuring agglutinating antibody levels against O and H antigens, this serological assay can provide evidence of recent or past typhoid infection. However, it is crucial to interpret Widal test results in conjunction with clinical findings and other laboratory tests. False positive results can occur due to cross-reactivity with other Enterobacteriaceae species or conditions like malaria and dengue. Conversely, false negatives may arise in early acute cases before antibodies develop or in chronic carriers with low antibody titers. Moreover, the Widal test cannot differentiate between active and previous infections nor distinguish the infecting Salmonella serotype. Its diagnostic utility is limited in endemic areas where baseline antibody titers tend to be elevated. While still widely used in resource-limited settings for presumptive diagnosis of typhoid, the Widal test has lower sensitivity and specificity compared to modern tests like blood/bone marrow cultures and nucleic acid amplification techniques. These improved methods allow for definitive detection and identification of the causative organism. When used judiciously and interpreted cautiously alongside other evidence, the Widal test remains a viable option for typhoid screening. However, its limitations highlight the need for improved diagnostics, especially rapid tests, to enhance typhoid surveillance and clinical management globally.

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