WIDAL TESTTalha RazaBsc (HONS) MLTFMH institute of allied health sciencesEmail: talharaza.mlt@gmail.com

INTRODUCTION OF WIDAL TEST Widal test is a serological test that is done for the

diagnosis of typhoid fever caused by Salmonella organism.

This test detects the “O” and “H” antigen of Salmonella typhi and paratyphi A,B and C.

When facilities for culturing are not available, the Widal test is the reliable and can be of value in the diagnosis of typhoid fevers in endemic areas. 

CLINICAL SIGNIFICANCE• Typhoid fever or enteric fever occurs when S. Typhi, S. Paratyphi

A, S. Paratyphi B, S. Paratyphi C infect the human body.• Body responds to this antigenic stuimulus by producing

antibodies whose titre rise slowly in early stages, to a maximum and then slowly falls till it is undetectable.

• Persons with typhoid fever carry the bacteria in their bloodstream and intestinal tract.

• Transmitted through the ingestion of food or drink contaminated by the feces or urine of infected people

TYPHOID FEVERHistory:• Thomas Willis who is credited with thefirst description of typhoid fever in 1659.

• French physician Pierre Charles Alexandre Louis first proposed

the name “typhoid fever”

CONT.The best known carrier was "Typhoid Mary“; Mary Mallon was a cook in Oyster Bay, New York in 1906 who is known to have infected 53 people, 5 of whom died.

2. Later returned with false name but detained and quarantined after another typhoid outbreak.

3. She died of pneumonia after 26 years in quarantine.

HOW DOES BACTERIA CAUSEIngestion of contaminated food or water

Salmonella bacteria

Invade small intestine and enter the bloodstream

Carried by white blood cells in the liver, spleen, and bone marrow

Multiply and reenter the bloodstream

CONT.Bacteria invade the gallbladder, biliary system, and the lymphatic

tissue of the bowel and multiply in high numbers

Then pass into the intestinal tract and can be identified for diagnosis in cultures from the stool tested in the laboratory

DIAGNOSISDiagnosis of typhoid fever is made by

• Blood culture• Stool culture• Bone marrow culture• Widal test• Typhidot (ICT/ELISA)• Antimicrobial susceptibility testing

HISTORY It was developed by Georges Ferdinand Widal in 1896.

PRINCIPLE

• “When the colored, smooth, killed Salmonella antigen suspensions are mixed/incubated with patient serum, anti salmonella antibody present in the patient serum react with the antigen suspensions to give agglutination. Agglutination is a positive test result, indicating presence of anti salmonella antibodies in the patient serum. No agglutination is a negative test result indicating absence of anti salmonella antibodies”.

• The patient’s serum is tested for O and H antibodies (agglutinins) against the following antigen suspensions (stained suspensions):

•

S. Typhi 0 antigen S. Typhi H antigen

• S. Paratyphi A H antigen S. Paratyphi B H antigen S. Paratyphi C H antigen

• The paratyphoid “O” antigen are not employed as they cross react with typhoid “O” antigen.

• “H” suspensions are colored red while “O” suspensions color is “Blue”.

Specimen Collection and Precautions

• No special preparation of the patient is required prior to sample collection.

• Don’t use hemolysed sample.• Don’t heat or inactivate the serum.• Freshly collected sample is a preferable, store at 2-8 C for up to 72 hours

in case of delay in testing.• It is preferable to test two specimens of sera at an interval of 7 to 10

days to demonstrate a rising antibody titer.• Salmonella antibody starts appearing in serum at the end of first week

and rise 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.

REQUIREMENTS Reagents commercially prepared antigen suspension of• S. typhi O (2-8°C)• S. typhi H (2-8°C)• S. paratyphi AH (2-8°C)• S. paratyphi BH (2-8°C)• Positive control (2-8°C)• Negative control (2-8°C) Glass slide/test card Disposable mixing sticks Test tubes

STORAGE:• Store all reagents at 2-8◦C.• Read carefully expiry date mentioned on kit before use.• Bring all reagents at room temperature, gently shake and mix

before use.

NOTE: • The S. Typhi “O” reagent contains 0.5 % phenol, S. Typhi

“H”, S. paratyphi “AH”, “BH” reagents contains 0.3 % formaldehyde as preservative.

• Avoid contact with skin and mucosa.

COMMERCIALLY PREPARED WIDAL TEST KIT

PROCEDURE1) Slide method2) Tube method

SLIDE METHOD QUALITATIVE METHOD Place one drop of positive control on one reaction circle of test card

or on slide. Pipette one drop of Normal saline on next circle for negative control. Pipette one drop of patient serum onto remaining four reaction

circles. Add one drop of H suspension on 1st and 2nd reaction circle (on +ve

and –ve control circle) Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the

remaining four reaction circles. Mix contents of each circle uniformly over the entire circle with

separate mixing sticks. Rotate the slide, gently back and forth and observe for agglutination

macroscopically within one minute.

AGGLUTINATION REACTION FOR DIFFERENT ANTIGEN SOLUTIONS WITH TEST SAMPLE

SEMI QUANTITATIVE METHOD Pipette 5, 10, 20, 40, 80 ul of the test sample on the reaction

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.

Serum volume Titre80 ul 1:2040 ul 1:4020 ul 1:8010 ul 1:16005 ul 1:320

STANDARD TUBE METHODTake four sets of 8 test tubes and label them 1 to 8 for O,H,AH and BH antibody detection. 

Pipette in to the 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 1ml of the diluted serum from tube no.1 to tube no.2 and mix well.

Discard the 1ml of the diluted serum from tube no.7 of each set.

Tube no.8 in all sets serves as a saline control. 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 tubes (1 to 8) of each set add one drop of the respective WIDAL TEST antigen suspension (O,H,AH,BH) from reagent vials and mix well.

Cover the tubes and incubate at 37 C overnight (approx. 18 hrs).

Dislodge the sedimented button gently and observe.

INTERPRETATION • Agglutination indicates positive test result.• Significant titre is >1:160 but varies from population to population.• Titre of “O” antigen more than 1:160 indicates recent infection.• Titre of “H” antigen more than 1:160 indicates past infection or due to

immunization.• False positive result may be in vaccination or previously infection with S.

typhi.• False negative may be due to antimicrobial treatment which blocks

antibody response.