Widal Test: Principle, Procedure, Results, Limitation

Widal test is the most widely used diagnostic test for typhoid fever in developing countries. The Widal test has been in use for more than a century as an aid in the diagnosis of typhoid fever. The Widal test is positive after the tenth day of the disease and may be falsely positive if an individual previously received a typhoid vaccine.

The Widal agglutination test is performed using a standardized suspension of S. enterica serotype Typhi ‘O’ and  ‘H’ and S. enterica serotype Paratyphi A ‘H’ and S. enterica serotype Paratyphi B ‘H’ antigen. It measures agglutinating antibody levels against O and H antigens using serial dilutions of sera. Tube agglutination method is the recommended method of performing the Widal test; where serial two-fold dilutions of the patient’s serum from 1:20 to 1:1280 are tested. After its development, the rapid slide test became the most commonly used technique in local laboratories because of its convenience.

Widal test was devised by Frank Widal in 1896. Widal originally described the test to diagnose Salmonella enterica serotype Paratyphi B infection.

Principle

Patients infected with S. enterica serotype Typhi and Paratyphi produce serum antibodies to the O and H antigens of these pathogens, and the detection of these specific antibodies forms the basis of Widal test.

Antigens specifically prepared from Salmonella are used in the Widal agglutination test to detect the presence of antibodies in patients’ serum. Four specific antigen suspensions are used in Widal test; they are O, H, AH, and BH. Unfortunately, S. enterica serotype Typhi shares these antigens with other Salmonella serotypes and shares cross-reacting epitopes with other Enterobacteriaceae which increases the chance of false-positive results.

The earliest serological response in acute typhoid fever is a rise in the titer of the O antibody, with an elevation of the H- antibody titer developing more slowly but persisting longer than that of the O- antibody. Usually, O antibodies appear on 6 to 8 days and H antibodies on 10-12 days after the onset of the disease. Patients from communities where typhoid is endemic have higher H-antibody titers so some researchers claim that the level of H agglutinins is unhelpful in the diagnosis of typhoid.

Materials required

• Widal test kit (killed colored suspension of S. enterica serotype Typhi O antigen, S. enterica serotype Typhi H antigen and S. enterica serotype Paratyphi AH antigen, and S. enterica serotype Paratyphi BH antigen).
• Incubator
• Normal saline
• Refrigerator
• Applicator stick
• Graduated pipette

Before use, bring all reagents to room temperature and mix well.

Procedure of Widal Test

Rapid screening test

1. Mark the circles of slides as PC (positive control), NC (negative control), O, H, AH, and BH
   as per antigen solutions used for testing.
2. Add 1 drop of positive control (25μL) into the circle marked as PC.
3. Then add 1 drop of negative control (25μL) into the circle marked as NC.
4. Add 1 drop of the test sample (25μL) into each circle labeled as O, H, AH, and BH.
5. Add 1 drop of antigen solution of Salmonella typhi ‘H’ into PC and NC circle each. Mix well by using a new mixing stick for each circle.
6. To circles labeled as O, H, AH, BH in which test samples have been added, add antigen solutions of Salmonella typhi ‘O’, Salmonella typhi ‘H’, Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’, respectively.
7. Mix the content of each reaction circle uniformly with a separate mixing stick.
8. Rock the glass slide gently (approximately for one minute) and observe for agglutination.

Result: 

• Positive Widal test: Agglutination was observed within a minute.
• Negative Widal test: No agglutination

Slide Titration

Rapid slide titration needs to perform for the samples which showed positive titer during rapid screening.

• Using a micropipette, dispense 40, 20, 10, and 5µl of undiluted serum onto a row of 3 cm diameter circles.
• Shake the reagent bottle rigorously shaken and add a drop (0.03 ml) of the undiluted antigen suspension to each serum aliquot.
• Mix it thoroughly mixed with the aid of a stirring stick and rotate the slide gently.
• Observe the reactions after a minute.

Result: 

• Positive test: Agglutination was observed within a minute.
• Negative test: No agglutination

Reporting Widal test

The Widal test is reported by giving the titer for both O and H antibodies. The titer of each serum is read as the highest serum dilution giving visible agglutination. The agglutination observed in any circle was indicative of the following results in a test tube.

Circle	1	2	3	4	5
Serum volume	80 µl	40 µl	20 µl	10 µl	5 µl
Amount of antigen	1 drop	1 drop	1 drop	1 drop	1 drop
Equivalent tube titre	1:20	1:40	1:80	1:160	1:320

Quality control

Quality control was done using the positive polyspecific control of the same dilutions as the test sample. Normal saline was used for negative control.

Importance of Baseline Titer in Widal Test

Ideally, the Widal test should be run on both acute and convalescent-phase sera to detect an increase in the agglutination titer but patient management cannot wait for results obtained with a convalescent-phase sample. For practical purposes, a treatment decision must be made on the basis of the results obtained with a single acute-phase sample. It is, therefore, important to establish the antibody level in the normal population in a particular locality in order to determine a threshold above which the antibody titer is considered significant.

In a situation where second sample collection is not feasible, knowledge of the agglutinin levels in the sera of normal subjects from the patients’ community can form the baseline on which a diagnosis can be made.

Interpretations of Widal Test

Although simple to perform, the Widal test is difficult to interpret, requiring detailed knowledge of the patient’s medical, travel, and vaccination history. The interpretation of a Widal test is greatly affected by the nature and extent of the patient’s previous contact with typhoid antigens, whether the contact depends on a clinical or subclinical infection with typhoid or related organisms or is from TAB vaccination. The less the degree of the previous contact, the greater the possibility that the findings of a Widal test may be usefully interpreted. In countries where typhoid is endemic, the Widal test can be interpreted only if the reporting laboratory has information about the basic level of O and H agglutinin in the population. Figure well in excess of the known titers especially if the amount of antibody rises during the illness are highly suggestive of infection.

Most of the researchers reported that Widal test has a diagnostic value when judged by clinical findings and knowledge of the baseline O and H agglutinin titers in the local population. Although there is no consensus on the diagnostic titer for a single Widal test, all the studies reported a “positive” Widal test based on a fourfold rise in O agglutinins in repeated tests or a titer of >1:80 or greater in a single test.

To curb the overdiagnosis and misdiagnosis of Typhoid fever, Adeleke and Ihesiulor, 2008 recommended a single Widal titer of >1:160 or a four-fold increase in titer between two samples taken at least 10 – 14 days apart, as diagnostic. Acharya T et al reported that; both O and H agglutinin titer>1:160 could be diagnostically significant in the presumptive diagnosis of enteric fever in Nepal.

Elevated levels of both O and H agglutinin titer are more helpful than either of them alone, in making a presumptive diagnosis of typhoid fever. When blood cultures are not available or impractical, a single Widal test can still have diagnostic significance, if the results are interpreted with relevant clinical findings and prevailing O and H agglutinin titers in the local population.

Limitations of Widal Test

The value of the Widal test in diagnosing enteric fever in endemic areas remains controversial but is still a useful and widely available test in endemic areas. Epidemiologic studies in an endemic country have shown that at least seven subclinical cases of typhoid fever occur for each clinical case. Therefore a positive Widal test may be seen in apparently healthy persons from an endemic area as a result of previous subclinical infection. 

• Previous typhoid vaccination may contribute to elevated agglutinins in the non-infected population.
• Cross reaction between malaria parasites and salmonella antigens may cause false-positive Widal agglutination test
• False-positive Widal tests have also been reported for patients with non-enteric salmonella infection, for example, typhus, immunological disorders, chronic liver disease, and cryptococcal meningitis.
• Prior use of antibiotics can dampen antibody response giving a low titer in the Widal test even in the face of bacteriologically confirmed typhoid fever resulting in misdiagnosis
• The Widal agglutination titer varies with the geographic location based on the endemicity of the enteric fever, the prevalence of non-typhoid salmonellae infection, and other infections which cross-react with salmonella antigen.
• Past infection with serotype Typhi or another nontyphoidal Salmonella serotype that shares common antigens gives a false positive Widal test.

Sensitivity and Specificity of Widal Test

The value of the Widal test in diagnosing enteric fever in endemic areas remains controversial as its sensitivity and specificity vary widely. Some experts suggest that the Widal test lacks standardization and adequate sensitivity and specificity to be clinically useful, while others consider the test to have diagnostic value when judged by clinical findings and knowledge of the ‘normal’ O and H agglutinin titers in the local population (‘baseline titers’).

Widal test is simple and inexpensive, so it has gained widespread use despite shortcomings of both sensitivity and specificity that compromise its utility as a diagnostic test.

Country	Agglutinin	Cut-off titer	Sensitivity (%)	Specificity (%)	Reference
Tube agglutination
Philippines	O	1:20	61	88	(Aquino et.al., 1991)
Philippines	O	1:80	64	100	(Buck et al., 1987)
Jordan	O and H	1:160	92	–	(Shehabi,1981)
Peru	O	1:160	58	–	(Levine, 1978)
H	1:160	82	–
Ceylon	O and H	1:160	85.7	88	(Senewiratne and Senewiratne, 1977)
Slide agglutination
Philippines	O	1:160	72.5	57.5	(Roxas et al., 1989)
O	1:320	57.5	100
Ethiopia	O and H	1:160	82	–	(Abraham et al., 981)
S.Africa	O and H	1:200	75	92.5	(Sommerville et al., 1981)

Salmonella Antigens

O Antigen

This is a somatic antigen and is present on the outer membrane of the bacteria. Its specificity is determined by the nature of the repeating units in the outer O-polysaccharide chain. Somatic antigens are heat stable, alcohol-resistant, and form 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 that is inactivated both by boiling and alcohol. H antigens rapidly form fluffy clumps when treated with the corresponding antisera. H antigen induces the rapid formation of corresponding antibodies as it is strongly immunogenic.

References

1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology). Lippincott Williams & Wilkins
2. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
3. Olopoenia, L. A., & King, A. L. (2000). Widal agglutination test – 100 years later: still plagued by controversy. Postgraduate medical journal, 76(892), 80–84. https://doi.org/10.1136/pmj.76.892.80 
4. Adhikari, A., Rauniyar, R., Raut, P. P., Manandhar, K. D., & Gupta, B. P. (2015). Evaluation of sensitivity and specificity of ELISA against Widal test for typhoid diagnosis in endemic population of Kathmandu. BMC infectious diseases, 15, 523. https://doi.org/10.1186/s12879-015-1248-6