Widal Test

 

Qualitative Widal Test for Detection of Salmonella Antibodies in Serum

Objective- To detect the presence of agglutinating antibodies (O and H antibodies) against Salmonella typhi and Salmonella paratyphi in a patient's serum sample to aid in the diagnosis of typhoid and paratyphoid fever.

Theory

The Widal test is a serological agglutination test that detects antibodies in the patient's serum directed against the O (somatic) and H (flagellar) antigens of Salmonella typhi and Salmonella paratyphi bacteria.

• O antigen: Heat-stable somatic antigen of Salmonella, indicating active infection.
• H antigen: Heat-labile flagellar antigen indicating current or past infection.

When a patient's serum containing antibodies is mixed with suspensions of killed Salmonella antigens, agglutination (clumping) occurs if antibodies specific to these antigens are present. The degree of agglutination, observed as clumping under a microscope or visually, indicates the antibody titer, helping in diagnosis.

Requirements / Materials

• Patient serum sample
• Standardized Salmonella typhi O and H antigen suspensions
• Standardized Salmonella paratyphi A and B antigen suspensions
• Normal saline (0.85% NaCl)
• Clean glass slides or test tubes
• Pipettes or micropipettes
• Positive and negative control sera

Procedure

1. Place a drop of the patient’s serum on a clean glass slide or test tube.
2. Add a drop of the respective Salmonella antigen suspension (O or H).
3. Mix gently with a clean applicator or pipette tip.
4. Observe the mixture for agglutination (clumping) within 1-2 minutes at room temperature.

Observation Table

Antigen Tested	Agglutination (+) / No Agglutination (–)
Salmonella typhi O antigen	+ / –
Salmonella typhi H antigen	+ / –
Salmonella paratyphi A O	+ / –
Salmonella paratyphi B O	+ / –

Result

• Positive: Visible clumping/agglutination in the mixture indicates the presence of antibodies against the tested antigen.
• Negative: No visible clumping indicates absence of detectable antibodies.

Discussion

The qualitative Widal test provides a quick indication of whether a patient has antibodies against Salmonella antigens, suggesting current or past infection. However, it does not measure the antibody level. Positive agglutination must be interpreted in the context of clinical symptoms and epidemiological factors. False positives may occur due to cross-reactivity, and false negatives may occur in early infection.

Conclusion

The qualitative Widal test is a simple, rapid screening test for typhoid and paratyphoid fever. Presence of agglutination suggests exposure or infection with Salmonella species, but further clinical and laboratory evaluation is recommended for confirmation.

Precautions

• Use standardized antigens to avoid false results.
• Avoid contamination of samples and reagents.
• Use clean glassware and pipettes for each test.
• Interpret results cautiously with clinical correlation.
• Include positive and negative controls to validate test results.

Reference

Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2. Cambridge University Press.