Widal Test : Principle, Procedure, and Result

                                      WIDAL TEST 

Introduction

Widal test is a serological method to diagnose enteric fever or typhoid which is caused by the infection with pathogenic microorganisms like Salmonella typhi, Salmonella paratyphi A, B, and C.

The method of this diagnostic test is based upon a visible agglutination reaction either in a test tube or on a slide between antibodies of patient serum and antigens specifically prepared from Salmonella species.

Principle : 

Enteric fever or typhoid is a life threatening disease usually occurs due to the infection of pathogenic microorganisms, e.g. Salmonella typhi, Salmonella paratyphi A, B, and C. These microorganisms are transmitted to human body through food and drinks contaminated with fecal matter.

Early diagnosis and treatment for this fever are essential to avoid serious clinical complications. During the course of infection antibodies are produced against Salmonella antigens.

The organisms causing enteric fever possesses two major antigens namely somatic antigen, O and a flagellar antigen, H along with another surface antigen, Vi. During infection antibodies are produced in patient's sera against Salmonella typhi O and H and Salmonella paratyphi AH and BH antigens. During infection antibodies are produced in patient's sera against these antigens.

Antigens specifically prepared from this organism are used in the agglutination test to detect the presence of antibodies in patient's sera which are elucidated in response to infection by these bacteria. 

These are some agglutinins that are produced in the patient's  serum during the fever period, which react with somatic antigen O of Salmonella typhi, A or B of Salmonella species.

In this test four species antigen suspensions are used e.g. H, Salmonella typhi (O antigen) , Salmonella paratyphi A and B. If agglutination occurs in A and B antigen then it is confirmed as positive for Salmonella paratyphi. Agglutination will occur in H antigen circle for all the cases of antigens like O, A, and B.  

What is O, Vi and H antigens ? 

O Antigen: This is a somatic antigen and is present on the outer membrane of the cell. Its specificity is determined by the nature of the repeating units in the outer O- polysaccharide chain. Somatic antigen are heat stable, alcohol resistant and forms compact and granular clumps when mixed with O antisera.

Vi Antigen: This is a virulence antigen which is a capsular polysaccharide that overlays the O antigen. This capsule is not necessary for infection but it increases the infectivity by making it less detectable by the body's immune system. It is heat labile and can be detected using Vi antisera. Vi antigen can interfere with O antigen testing.

H Antigen: This is a heat labile flagellar antigen which is inactivated both by boiling and alcohol. H antigen rapidly form fluffy clumps when treated with the corresponding antisera. H antigen induces rapid formation of corresponding antibodies as it is strongly immunogenic.

How to perform widal test ?

• Before starting the experiment, bring all reagents to room temperature and mix well .
• Mark the circle of slide as PC ( positive control), NC (negative control), O,H,AH,and BH as per antigen solutions used for testing.
• Add 1-1 drop of positive control and negative control into the marked PC and NC respectively.
• Add 1 drop of test sample into each reaction circle labelled as O,H,AH, BH.
• Add 1drop of antigen solution of Salmonella typhi 'H' into PC and NC . Mix well with using new mixing stick for each circle.
• To circles labelled as O, H, AH and BH in which test samples has been added, add antigen solutions of Salmonella typhi'O' , Salmonella typhi'H' , Salmonella paratyphi'AH' and Salmonella paratyphi'BH' respectively.
• Mix the content of each reaction circle uniform with separate mixing stick.
• Rock the glass slide gently and observe for agglutination.

Result Interpretation 

Positive: 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 titres are present. S typhi O positive means active infection of typhoid fever. Active infection indicates the patient has an infection of Salmonella enterica serovar typhi. S typhi H positive means there is a past infection or it shows the result of the immunized person’s serum test report.

Negative: Widal test negative means that a person does not have enteric fever, and there is another infection causing the symptoms. The titre value of antigen O and H is below 1:160 in the sample.

Important questions asked.

What is the Widal test normal range chart?

When the test report lies in the Widal test normal range chart, then it is negative for the typhoid fever. If the titre value is less than or equal to 1:20, 1:40, 1:80, and less than 1:160 in the test report, then the typhoid test result lies in Widal test normal value.