Widal test

Principle

• Agglutination test detecting antibodies in patient serum against Salmonella antigens: S. Typhi O (somatic) and H (flagellar) antigens, and S. Paratyphi A and B H antigens (AH, BH); some kits include AO and BO.
• O agglutination: fine, compact, granular (chalky) clumps. H agglutination: large, loose, fluffy (cotton-wool) floccules.
• O antibodies (mainly IgM) appear earlier and decline sooner; H antibodies persist longer, especially after vaccination.

Specimen

• Serum; ideally paired acute and convalescent samples taken 7–10 days apart. Antibodies appear toward the end of the first week of fever.

Procedure

• Slide test (screening): fixed decreasing serum volumes (e.g. 80, 40, 20, 10, 5 µL) with one drop of antigen, approximating titres of 1:20–1:320.
• Tube test (standard, quantitative): doubling dilutions of serum (e.g. 1:20–1:640) with each antigen suspension; incubate per kit (commonly 37 °C overnight) and read for agglutination.
• Include positive and negative controls and a saline (antigen) control.

Interpretation

• Titre: highest dilution showing visible agglutination.
• A fourfold rise in titre between paired sera is the most reliable evidence of current infection.
• Single-sample cut-offs depend on local baseline titres; in many endemic areas O and H titres of ≥1:160 are considered significant. Local cut-offs apply.

Quality control and pitfalls

• False positives: previous typhoid vaccination (mainly raised H), past infection, anamnestic response in other febrile illnesses (malaria, dengue), cross-reacting Salmonella and other Enterobacterales, chronic liver disease.
• False negatives: early disease (first week), prior antibiotics, immunosuppression, carriers, prozone.
• Low sensitivity and specificity; results must be interpreted with clinical findings.

Clinical use

• Supportive test for enteric fever. Blood culture (first week) remains the diagnostic method of choice; bone marrow culture is most sensitive.