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 Agglutination Test
Agglutination reactions of O, H, AH, and BH antigen in Widal test

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.

Widal Test: Sample showing H positive in screening test
Widal Test: Sample showing H positive in screening test

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
Widal Test Result: Sample (6) showing BH positive in screening test
Widal Test Result: Sample (6) showing BH positive in screening test

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

Widal Test: Quantitative test for H antibody (Serial double dilution order from 1-2-3-6-5)
Widal Test: Quantitative test for H antibody (Serial double dilution order from 1-2-3-6-5)

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.

Circle12345
Serum volume80 µl40 µl20 µl10 µl5 µl
Amount of antigen1 drop1 drop1 drop1 drop1 drop
Equivalent tube titre1:201:401:801:1601:320

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.

CountryAgglutininCut-off titerSensitivity (%)Specificity (%)Reference
Tube agglutination
PhilippinesO1:206188(Aquino et.al., 1991)
PhilippinesO1:8064100(Buck et al., 1987)
JordanO and H1:16092(Shehabi,1981)
PeruO1:16058(Levine, 1978)
H1:16082
CeylonO and H1:16085.788(Senewiratne and Senewiratne, 1977)
Slide agglutination
PhilippinesO1:16072.557.5(Roxas et al., 1989)
O1:32057.5100
EthiopiaO and H1:16082(Abraham et al., 981)
S.AfricaO and H1:2007592.5(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

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

23 thoughts on “Widal Test: Principle, Procedure, Results, Limitation

  1. Fever is 102 F persistent ,more than 2 weeks, Typhi H antigen is + 1/80 ,but others widal antigens are negative. Clinically she looks like typhoid fever.Is this positive H antigen test is significant for typhoid fever?

    1. Dear Dr. Htun
      Thank you so much for your question. We can not rely on single H titre of 1/80 for the diagnosis. It might be because of number of conditions such as previous sub clinical infection, vaccination, other Enterobacteriace antigen etc (please correlate accordingly). Persistent fever of 102F is also not specific to make diagnosis. So if there is no other clinical signs of Typhoid fever, it may not be Typhoid. Before making confirmation, i will suggest two laboratory test. 1. If you can perform the culture, please do culture 2. If culture facility is not available, please repeat Widal test again, if infection there must be 4 fold rise in titre.

  2. Sir, Widal test results are:s typhi o:1:80 dilutions, s typhi h:1:160 dilutions.
    sp typhi ah and sp typhi bh:nonreacive. No syptoms of typhiod except 100 fever cough cold. then am i suffering from typhoid fever.

    1. Desu,
      First of all, i request you to visit clinician, he/she will evaluate your signs and sympotms and may order appropriate test, based on the duration of fever/symptoms etc. Remember that we develop “O” and “H” antibodies because of vaccination or previous sub-clinical infections by Salmonella or other members of Enterobacteriaceae family, which shares similar antigens. So presence of “O” and “H” titre may be because of this. To find out it is signficant or not, we need information regarding baseline titre “O” and “H” titre in your are and endemicity of the disease. If the disease is endemic in your area, it is likely to be because of previous contacts and you may not be suffering from Enteric fever but from some other infections (viral or parasitic or bacterial). Again, i request you to visit hospital for further work out and does not rely only on Widal Test.

  3. Sir
    Widal test results are: s typhi o – 1: 500 & s typhi h – 1:250.
    S para Ah-1:50 & Bh- 1: 50
    Please tell me, am i suffering from typhiod

    1. Lekh Ram Chaudhary sir, its hard to interpret your widal test results without knowing about your history and prevailing baseline titre of O and H in your locality. I request you to visit nearby hospital or consult with attending physician regarding your Widal test result. Please remember, Widal test gives idea about Antibodies formed in your body against Salmonella or related bacteria. That antibody can be formed by vaccination (if you have had) or previous sub clinial infections with any related genera or previous infection with Salmonella (if you had one). More definite test to know typhoid infection is blood culture. Please consult with physician once.

    2. Sisal test .S.tyhi o, H 1/80. 1/160 and s.paratyphi AH ,BH 1/20. 1/20/DL COMPARE HIGH OR LOW REPLY

      1. Dr. Sumit you have to interpret the results of Widal test based on the prevailing Basline Titre in that particular locality and his/her current presentations etc.

  4. Sir i have a question we hav o and h antigen in blood if we infect with thypoid and we develop antibody against it.And the antibody reacts with antigen . In the blood itself then why not we see any agglutination in the blood.?

    1. Gopi Ji, if Antigen-Antibody reacts in the blood Ag-Ab complex is formed. Which has various fates depending on various situations, such as opsonization, complement fixation, phagocytosis and sometimes it may deposit in various places, causing hypersensitivity reaction too.

  5. Widal slide test result is
    To – +ve , TH- negative, AH- negative, BH- negative
    What mean that result and what is the normal range and what is affected.

  6. Thanks for the comprehensive information on the Widal test and its interpretation. I want determine the baseline titre of O and H inmy community, please how do I go about the process?

    1. It might be normal/baseline titre in your locality. Please consult with your physician

  7. widal result S. O -ve S. H 1/80 S. O para A 1/80 S. O para B 1/160 S. H para A 1/80
    female 29 years pregnant for 6 month

    1. Dear Rania
      The widal titre differs according to the endemicity of the Enteric fever in your locality (so does the baseline titre; titre that a normal/healthy individuals may have). Its better to consult with your physician, they will have both physical examination, other check ups if necessary and can interpret this result incorporating all those findings too.

  8. Hi sir,yesterday I did widal test to my kid but esr increased,what is esr,why come to this problom

  9. The leucocyte count is 15300 and the widal test is positive O-1:80, H-1:160, the report is correct or not

  10. sir widal test reports are O 1:160 ,H 1:80 ,AH nil BH nil , result- weak negative.sir what does it means .i previously infected by typhoid previous year at the same seasons.

    symptoms- few few days before sneezing, nose flowing,mild fever

  11. Since my undergraduation and post graduation, interpretation of widal had been always a matter of debate. so kindly lease elaborate importance of each antigen when to consider it positive or negative.
    Like I have case of fever high grade not responding to anti pyeretics, no focus of infection, mild diarhoea, mild cough mild abdominal pain , Widal only revealed antigen H positive above 320 titre.

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