Widal Test is also known as Warm agglutinins; Agglutinins an agglutination test that detects the presence of serum agglutinins (H and O) in patients’ serum with typhoid and paratyphoid fever. When facilities for culturing are not available, the Widal test is reliable and can be of value in the diagnosis of typhoid fevers in endemic areas. Widal test positive values indicate that the person has the Salmonella enterica serovar Typhi bacterial infection. In the typhoid report positive, both O and H antigen titers are present. S typhi O positive means active infection of typhoid fever. In the case of the singular Widal test, baseline values for the normal range were found to be 1:20 – 1:80 for all the antigens (TO, TH, AO, AH, BO, BH), except BH, for which it was 1:20-1:40.
Agglutinins are antibodies that cause the red blood cells to clump together.
- Cold agglutinins are active at cold temperatures.
- Febrile (warm) agglutinins are active at normal body temperatures.
This article describes the blood test that is used to measure the level of these antibodies in the blood.
How the Test is Performed
A blood sample is needed .
How to Prepare for the Test
There is no special preparation.
How the Test will Feel
When the needle is inserted to draw blood, some people feel moderate pain. Others feel only a prick or stinging. Afterward, there may be some throbbing where the needle was inserted.
Why the Test is Performed
This test is done to diagnose certain infections and find the cause of hemolytic anemia (a type of anemia that occurs when red blood cells are destroyed). Knowing whether there are warm or cold agglutinins can help explain why hemolytic anemia is occurring and direct treatment.
Normal Results
Normal results are:
- Warm agglutinins: no agglutination in titers at or below 1:80
- Cold agglutinins: no agglutination in titers at or below 1:16
The examples above are common measurements for the results of these tests. Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or test different samples. Talk to your doctor about the meaning of your specific test results.
What Abnormal Results Mean
An abnormal (positive) result means there were agglutinins in your blood sample.
Warm agglutinins may occur with:
- Infections, including brucellosis , rickettsial disease , salmonella infection , and tularemia
- Inflammatory bowel disease
- Lymphoma
- Systemic lupus erythematosus
- Use of certain medicines, including methyldopa, penicillin, and quinidine
Cold agglutinins may occur with:
- Infections, especially mononucleosis and mycoplasma pneumonia
- Chickenpox (varicella)
- Cytomegalovirus infection
- Cancer, including lymphoma and multiple myeloma
- Listeria monocytogenes
- Systemic lupus erythematosus
- Waldenström macrogolulinemia
Risks
Risks are slight but may include:
- Excessive bleeding
- Fainting or feeling lightheaded
- Hematoma (blood accumulating under the skin)
- Infection (a slight risk any time the skin is broken)
Considerations
If a disease linked to cold agglutinin is suspected, the person needs to be kept warm
- The patient’s serum is tested for O and H antibodies (agglutinins) against the following antigen suspensions (usually stained suspensions):
S. Typhi 0 antigen suspension, 9, 12
S. Typhi H antigen suspension, d
S. Paratyphi A 0 antigen suspension, 1, 2, 12
S. Paratyphi A H antigen suspension, a
S. Paratyphi B 0 antigen suspension, 1, 4, 5, 12
S. Paratyphi B H antigen suspension, b, phase 1
S. Paratyphi C 0 antigen suspension, 6, 7
S. Paratyphi C H antigen suspension, c, phase 1 - Salmonella antibody starts appearing in serum at the end of the first week and rises sharply during the 3rd week of endemic fever. In acute typhoid fever, O agglutinins can usually be detected 6–8 days after the onset of fever and H agglutinins after 10–12 days.
- It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a rising antibody titer.
- Salmonella antigen suspensions can be used as slide and tube techniques.
Principle of Widal Test
The bacterial suspension which carries antigen will agglutinate on exposure to antibodies to Salmonella organisms. Patients suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial doubling dilutions of killed, colored Salmonella antigens in an agglutination test.
The main principle of the Widal test is that if the homologous antibody is present in the patient’s serum, it will react with the respective antigen in the reagent and gives visible clumping on the test card and agglutination in the tube. The antigens used in the test are “H” and “O” antigens of Salmonella Typhi and “H” antigens of S. Paratyphi. The paratyphoid “O” antigen is not employed as they cross-react with typhoid “O” antigen due to the sharing of factor 12. “O” antigen is a somatic antigen and “H” antigen is flagellar antigen.
Preparation of Widal Antigens
- H suspension of bacteria is prepared by adding 0.1 percent formalin to a 24 hours broth culture or saline suspension of an agar culture.
- For the preparation of O suspensions of bacteria, the organisms is cultured on phenol agar (1:800) to inhibit flagella.
- Standard smooth strains of the organism are used; S Typhi 901, O, and H strains are employed for this purpose.
- The growth is then emulsified in a small volume of saline, mixed with 20 times its volume of alcohol, heated at 40° C to 50° C for 30 minutes, and centrifuged.
- The antigens are treated with chloroform (preservative) and appropriate dyes are added for easy identification of antigens.
The procedure of the Widal Test
SLIDE TEST
- Place one drop of positive control on one reaction circle of the slide.
- Pipette one drop of Isotonic saline on the next reaction circle. (-ve Control).
- Pipette one drop of the patient serum to be tested onto the remaining four reaction circles.
- Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction circles. (PC & NC).
- Add one drop each of ‘O’, ‘H’, ‘AH’, and ‘BH’ antigens to the remaining four reaction circles.
- Mix the contents of each circle uniformly over the entire circle with separate mixing sticks.
- Rock the slide, gently back and forth, and observe for agglutination macroscopically within one minute.
SEMI-QUANTITATIVE METHOD
- Pipette one drop of isotonic saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul of the test sample on the remaining circles.
- Add to each reaction circle, a drop of the antigen which showed agglutination with the test sample in the screening method.
- Using separate mixing sticks, mix the contents of each circle uniformly over the reaction circles.
- Rock the slide gently back and forth, observe for agglutination macroscopically within one minute.
STANDARD TUBE TEST METHOD
In Widal Test, two types of tubes were originally used:
(1) Dreyer’s tube (a narrow tube with conical bottom) for H agglutination and
(2) Felix tube (short round-bottomed tube) for O agglutination.
Nowadays 3 x 0.5 ml Kahn tubes are used for both types of agglutination.
- Take 4 sets of 8 Kahn tubes/test tubes and label them 1 to 8 for O, H, AH, and BH antibody detection.
- Pipette into tube No.1 of all sets 1.9 ml of isotonic saline.
- To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
- To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested and mix well.
- Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
- Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix well. Continue this serial dilution till tube no.7 in each set.
- Discard 1.0 ml of the diluted serum from tube No.7 of each set.
- Tube No.8 in all the sets, serves as a saline control. Now the dilution of the serum sample achieved in each set is as follows: Tube No. : 1 2 3 4 5 6 7 8 (control) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280.
- To all the tubes (1 to 8) of each set add one drop of the respective WIDALTEST antigen suspension (O, H, AH, and BH) from the reagent vials and mix well.
- Cover the tubes and incubate at 37° C overnight (approximately 18 hours).
- Dislodge the sedimented button gently and observe for agglutination.
The above protocols are obtained from WIDAL TEST: Seemed Diagnostics
Interpretation of Widal Test
SLIDE TEST
- Agglutination is a positive test result and if the positive reaction is observed with 20 ul of the test sample, it indicates the presence of clinically significant levels of the corresponding antibody in the patient serum.
- No agglutination is a negative test result and indicates the absence of clinically significant levels of the corresponding antibody in the patient serum.
TUBE-METHOD
- The titer of the patient serum using Widal test antigen suspensions is the highest dilution of the serum sample that gives a visible agglutination.
- The sample which shows the titer of 1:100 or more for O agglutinations and 1:200 or more for H agglutination should be considered clinically significant (active infection). Example: In the above figure, the titer is 160.
- Demonstration of a 4-fold rise between the two is diagnostic.
- H agglutination is more reliable than O agglutinin.
- Agglutinin starts appearing in serum by the end of 1st week with a sharp rise in the 2nd and 3rd weeks and the titer remains steady till 4th week after which it declines.
Limitations of Widal Test
- The Widal test is time-consuming (to find antibody titer) and often times when the diagnosis is reached it is too late to start an antibiotic regimen.
- The Widal test should be interpreted in the light of baseline titers in a healthy local population.
- The Widal test may be falsely positive in patients who have had previous vaccination or infection with S. Typhi.
- Besides cross-reactivity with other Salmonella species, the test cannot distinguish between current infection and previous infection or vaccination against typhoid.
- Widal titers have also been reported in association with the hypogammaglobulinemia of chronic active hepatitis and other autoimmune diseases.
- False-positive Widal test results are also known to occur in typhus, acute falciparum malaria
(particularly in children), chronic liver disease is associated with raised globulin levels and disorders such as rheumatoid arthritis, myelomatosis, and nephrotic syndrome. - False-negative results may be associated with early treatment, with “hidden organisms” in bone and joints, and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic.
- False-negative Widal tests may be due to antibody responses being blocked by early antimicrobial treatment or following a typhoid relapse.
- Severe hypoproteinaemia may also prevent a rise in 0 and H antibody titers.
- The antibody levels found in a healthy population, however, may vary from time to time and in different areas, making it difficult to establish a cut-off level of baseline antibody in a defined area and community.
- In low typhoid endemic areas, weak and delayed O and H antibody responses limit the usefulness of the Widal test. Variations also exist between laboratories in the performance and reading of Widal tests which compromise further the reliability of the test.
- The World Health Organization (WHO) has said that due to the various factors that can influence the results of a Widal test, it is best not to rely too much on this test.
